Method for producing recombinant adeno-associated virus particles

Abstract

The present invention addresses the problem of providing a novel method for producing rAAV particles, the method being capable of individually controlling the preparation steps for a capsid, a Rep protein, and a DNA fragment and controlling DNA encapsulation reaction conditions. The problem is solved by a method for producing recombinant adeno-associated virus particles, the method comprising a step for mixing a capsid synthesized in a first system, a Rep protein synthesized in a second system different from the first system, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid.

Description

Method for producing recombinant adeno-associated virus particles

[0001] The present invention relates to a method for producing recombinant adeno-associated virus particles, recombinant adeno-associated virus particles, and compositions that can be used in the method for producing recombinant adeno-associated virus particles.

[0002] Adeno-associated virus (AAV) is a linear, single-stranded DNA virus belonging to the Parvoviridae family. This virus is non-pathogenic, and its application in gene therapy is being investigated.

[0003] AAV particles have a structure in which the AAV genome is enclosed by a capsid. Terminal inversion repeats (ITRs) are present at both ends of the AAV genome and function as primers necessary for replication. Rep and cap genes are located between the ITRs. Each of these genes codes for multiple open reading frames (ORFs). The rep gene codes for the Rep protein, which has DNA helicase activity, ATPase activity, and nicking activity that introduces single-strand breaks (nicks) into double-stranded DNA, and is essential for the inclusion of the AAV genome in the capsid. The cap genes code for structural proteins that make up the capsid, called VP1, VP2, and VP3.

[0004] When AAV infects a host, it replicates in the presence of helper viruses such as adenoviruses and herpes simplex viruses. Conversely, in the absence of helper viruses, the AAV genome does not replicate and is instead integrated into the AAVS1 region located on human chromosome 19. It is known that the rep gene is required for integration into the AAVS1 region.

[0005] In gene therapy applications, recombinant adeno-associated virus (rAAV) particles are used, which package two ITRs (Integrated Transmutation Reactors) with the target gene inserted between them. To produce rAAV particles, cells are transfected with two plasmids: a GOI plasmid containing the target gene (Gene of Interest: GOI) encapsulated in a capsid, and a Rep / Cap plasmid containing the rep gene and the cap gene. Furthermore, cells are infected with a helper virus to replicate the target gene (for example, Patent Document 1). In recent years, since the genes responsible for the helper function of helper viruses (also called helper genes) have been identified, a method has been adopted in which three plasmids—the two plasmids mentioned above plus an adenovirus helper plasmid containing the helper gene—are simultaneously transfected into cells without infecting them with a helper virus (for example, Patent Document 2). Furthermore, cell lines have been modified to stably express the rep gene, cap gene, and helper gene by incorporating them into the genome (Patent Document 3).

[0006] Japanese Patent Publication No. 11-512923, Japanese Patent Publication No. 2021-510496, Japanese Patent Publication No. 2024-144435

[0007] In the methods described in Patent Documents 1 to 3, the capsid and Rep protein are expressed in the same cell, and the DNA fragment encoding the target gene contained in the AAV vector is encapsulated in the capsid within the cell. Therefore, it was difficult to individually control the preparation steps of the capsid, Rep protein, and DNA fragment, or to control the DNA encapsulation reaction conditions. In view of the above problems, the object of the present invention is to provide a novel method for producing rAAV particles that allows for individual control of the preparation steps of the capsid, Rep protein, and DNA fragment, and control of the DNA encapsulation reaction conditions.

[0008] To individually control the preparation steps of the capsid, Rep protein, and DNA fragment, and to control the conditions of the DNA encapsulation reaction, it is conceivable to purify the necessary factors such as the capsid and Rep protein and react them in a cell-free system. On the other hand, since many factors derived from host cells and helper viruses are also thought to be necessary for the formation of rAAV particles, research into encapsulating DNA in capsids in a cell-free system has not progressed. As a result of diligent research to solve the above problem, the inventors of this invention have found that rAAV particles can be produced by mixing the capsid and Rep protein with a polynucleotide encoding the target sequence, and have completed the present invention.

[0009] In other words, the present invention can be illustrated as follows: [1] A method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid synthesized in a first system, a Rep protein synthesized in a second system different from the first system, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid. [2] A method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid, wherein the composition produced by the mixing in the step comprises substantially only a capsid and a Rep protein as proteins. [3] A method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid, wherein the composition produced by the mixing in the step does not contain E1A, E1B, E2A, E4, and VA. [4] The method for producing the product according to any one of [1] to [3], wherein the capsid is purified. [5] The method for producing the product according to any one of [1] to [4], wherein the Rep protein is purified. [6] The method for producing the product according to any one of [1] to [4], wherein the polynucleotide is linear. [7] The method for producing the product according to [6], wherein the polynucleotide is linear and double-stranded. [8] The method for producing the product according to any one of [1] to [6], wherein the length of the polynucleotide is 20 to 5,000 bases. [9] The method for producing the product according to any one of [1] to [8], wherein the polynucleotide contains a modified residue.

[10] The method for producing the product according to any one of [1] to [9], wherein the polynucleotide does not contain part or all of the ITR.

[11] The method for producing the product according to any one of [1] to

[10] , wherein the polynucleotide contains RNA.

[12] The method for producing the product according to [1], wherein the first system uses a first cell that expresses the capsid.

[13] The manufacturing method according to [1] or

[12] , wherein the second system uses a second cell that expresses the Rep protein.

[14] The manufacturing method according to any one of [1] to

[13] , wherein the Rep protein is one or more selected from Rep78, Rep68, Rep52, and Rep40.

[15] The manufacturing method according to any one of [1] to

[14] , wherein the Rep protein is one or more selected from Rep78 and Rep68.

[16] The manufacturing method according to any one of [1] to

[15] , wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 1 pM or more and 1 μM or less.

[17] The manufacturing method according to any one of [1] to

[16] , wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 10 pM or more and 100 nM or less.

[18] The manufacturing method according to any one of [1] to

[17] , wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 100 pM or more and 50 nM or less.

[19] The manufacturing method according to any one of [1] to

[18] , wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 1 nM or more and 10 nM or less.

[20] The manufacturing method according to any one of [1] to

[19] , wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 1 pM or more and 100 μM or less.

[21] The manufacturing method according to any one of [1] to

[20] , wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 10 pM or more and 10 μM or less.

[22] The manufacturing method according to any one of [1] to

[21] , wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 1 nM or more and 1 μM or less.

[23] The manufacturing method according to any one of [1] to

[22] , wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 10 nM or more and 100 nM or less.

[24] The manufacturing method according to any one of [1] to

[23] , wherein in the encapsulation step, the polynucleotide is mixed so that the concentration of the polynucleotide is 1 pM or more and 1 μM or less.

[25] The manufacturing method according to any one of [1] to

[24] , wherein in the encapsulation step, the polynucleotide is mixed so that the concentration of the polynucleotide is 10 pM or more and 100 nM or less.

[26] The manufacturing method according to any one of [1] to

[25] , wherein in the encapsulation step, the polynucleotide is mixed so that the concentration of the polynucleotide is 100 pM or more and 20 nM or less.

[27] The manufacturing method according to any one of [1] to

[26] , wherein in the encapsulation step, the polynucleotide is mixed so that the concentration of the polynucleotide is 1 nM or more and 20 nM or less.

[28] The manufacturing method according to any one of [1] to

[27] , wherein in the encapsulation step, a nonionic water-soluble polymer is further mixed.

[29] The manufacturing method according to

[28] , wherein the weight-average molecular weight of the water-soluble polymer is 600 to 4,000,000.

[30] The method for producing a water-soluble polymer according to

[28] or

[29] , wherein the water-soluble polymer is one or more selected from the group consisting of polyether-based water-soluble polymers, water-soluble polysaccharides, vinyl-based water-soluble polymers, and sucrose-epichlorohydrin copolymers.

[31] The method for producing a water-soluble polymer according to any one of

[28] to

[30] , wherein the water-soluble polymer is one or more selected from the group consisting of polyethylene glycol (PEG), poly(propylene glycol), dextran, methylcellulose, polyvinylpyrrolidone, and sucrose-epichlorohydrin copolymers.

[32] The method for producing a water-soluble polymer according to any one of

[28] to

[31] , wherein in the encapsulation step, the water-soluble polymer is mixed so that the concentration of the water-soluble polymer is 0.03 to 10% (w / v).

[33] The method for producing a water-soluble polymer according to any one of [1] to

[32] , wherein in the encapsulation step, a surfactant is further mixed.

[34] The method for producing a water-soluble polymer according to

[33] , wherein the surfactant is a nonionic surfactant.

[35] The manufacturing method according to

[33] or

[34] , wherein the surfactant has a polyoxyethylene chain.

[36] The manufacturing method according to any one of

[33] to

[35] , wherein in the encapsulation step, the surfactant is mixed so that the concentration of the surfactant is 0.0001 to 3% (w / v).

[37] A method for manufacturing according to any one of [1] to

[36] , wherein the encapsulation step is performed at 20 to 55°C.

[38] A method for manufacturing according to [2], wherein the composition does not contain E1A, E1B, E2A, E4, and VA.

[39] A method for manufacturing according to [2], [3], or

[38] , wherein the composition does not substantially contain cell lysate.

[40] A recombinant adeno-associated virus particle in which a) a modified residue is contained, b) part or all of the ITR is not contained, or c) a polynucleotide containing RNA is encapsulated in the capsid.

[41] A recombinant adeno-associated virus particle according to

[40] , wherein the polynucleotide is linear.

[42] A recombinant adeno-associated virus particle according to

[40] or

[41] , wherein the polynucleotide contains a modified residue.

[43] A composition comprising a capsid, a Rep protein, and a polynucleotide comprising a) a modified residue, b) a part or all of an ITR, or c) RNA, and substantially free from cell lysate.

[44] The composition according to

[43] , wherein the capsid is purified.

[45] The composition according to

[43] or

[44] , wherein the Rep protein is purified.

[46] The composition according to any one of

[43] to

[45] , wherein the Rep protein is one or more selected from Rep78, Rep68, Rep52, and Rep40.

[47] The composition according to any one of

[43] to

[46] , wherein the polynucleotide is linear.

[48] The composition according to any one of

[43] to

[47] , wherein the polynucleotide comprises a modified residue.

[0010] This invention allows for the individual control of the preparation steps for the capsid, Rep protein, and DNA fragment in the production of rAAV particles. Furthermore, it enables control of the DNA encapsulation reaction conditions. Additionally, it makes it possible to produce rAAV particles containing polynucleotides that could not be encapsulated using conventional methods for producing rAAV particles in host cells.

[0011] This graph shows the concentration (vg / μL) of the vector sequence encapsulated in the AAV2-derived capsid. This graph shows the concentration (vg / μL) of each sequence encapsulated in the AAV2-derived capsid. This graph shows the concentration (vg / μL) of the ITR-containing sequence encapsulated in the AAV1, AAV2, or AAV6-derived capsid. This graph shows the concentration (vg / μL) of each DNA fragment containing modified residues encapsulated in the AAV2-derived capsid. This graph shows the concentration (vg / μL) of the encapsulated sequence encapsulated in the capsid in Experimental Example 6. This graph shows the concentration (vg / μL) of the encapsulated sequence encapsulated in the capsid in Experimental Example 7. This graph shows the results of mass photometry measurement. This graph shows the concentration (vg / μL) of the encapsulated sequence encapsulated in the capsid in Experimental Example 10. This graph shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid in Experimental Example 11. This graph shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid when the total monovalent cation concentration is changed under conditions without the addition of PEG6000. This graph shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid when the total monovalent cation concentration is changed under conditions with the addition of 3% PEG6000. This graph shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid when the PEG6000 concentration and the total monovalent cation concentration are changed under conditions with and without the addition of Rep68. This graph shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid when the type and concentration of surfactant are changed. This graph shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid in the hypotonic buffer fraction or the storage buffer fraction when the inclusion reaction was performed under conditions with / without lysated cells and helper factors. This graph shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid when sequences 1 to 7 with different sequence lengths were used as inclusion sequences. This graph shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid when sequences 1, 8 to 13 with different sequence lengths were used as inclusion sequences.This is a graph showing the concentration (vg / µL) of inserted sequences encapsidated in the capsid when using wild-type Rep68, Y156F mutant, or K340H mutant. It is also a graph showing the concentration (vg / µL) of inserted sequences encapsidated in the capsid when the temperature during the encapsidation reaction is changed.

[0012] Hereinafter, the present invention will be described in detail with reference to embodiments, examples and the like. However, the present invention is not limited to the following embodiments, examples and the like, and can be arbitrarily modified and implemented without departing from the scope of the gist of the present invention. In the present specification, a numerical range represented by using "~" means a range including the numerical values before and after "~" as the lower limit and the upper limit, and "A to B" means from A to B (inclusive).

[0013] One embodiment of the present invention is a method for producing recombinant adeno-associated virus particles, comprising the step of mixing a capsid synthesized in a first system, a Rep protein synthesized in a second system different from the first system, and a polynucleotide encoding a target sequence, and allowing the capsid to encapsidate the polynucleotide. In the present specification, the term "recombinant adeno-associated virus (rAAV) particle" refers to a particle in which a polynucleotide is encapsulated in a capsid. In the present specification, rAAV particles are also referred to as full capsids.

[0014] <Capsid> The capsid used in the present embodiment is synthesized in a first system. The first system may use first cells that express the capsid. That is, the production method of the present embodiment may include a step of expressing the capsid in the first cells. In addition, the first system may use a cell-free synthesis system. That is, the production method of the present embodiment may include a step of synthesizing the capsid using a cell-free synthesis system. Commercially available capsids may also be used.

[0015] The first cell used in the step of expressing a capsid in the first cell is not particularly limited as long as it is capable of expressing a capsid. Examples of cells include bacteria, actinomycetes, yeasts, fungi, plant cells, insect cells, and mammalian cells. Examples of bacteria include coryneform bacteria such as Escherichia coli and Corynebacterium glutamicum. Examples of yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastris. Examples of plant cells include tobacco. Examples of insect cells include Sf9 cells. Examples of mammalian cells include human cells such as HEK293 cells, CHO cells, and Hela cells. Among these, HEK293 cells, Saccharomyces cerevisiae, CHO cells, Sf9 cells, tobacco, Schizosaccharomyces pombe, or Pichia pastris are preferred.

[0016] The capsid is composed of multiple capsid proteins, typically consisting of VP1, VP2, and VP3. In this embodiment, the capsid preferably contains VP1, VP2, and / or VP3, and more preferably contains VP1, VP2, and VP3. These capsid proteins self-assemble in solution to form a shell-like structure capable of encapsulating polynucleotides. This structure includes common folds, including a β-barrel structure, subunit interaction patterns, and structural elements related to the shape of the particle lumen, and these structural elements are known to be highly conserved among serotypes. Hereinafter, structures containing these highly conserved structural elements may be referred to as conserved structures. In this specification, a capsid that does not contain polynucleotides may be referred to as an "empty capsid." The capsid used in the production of recombinant adeno-associated virus particles in the present invention may be an empty capsid.

[0017] The capsid may be derived from any AAV, specifically AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, or AAVrh10, with AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, or AAV9 being preferred. It may also be a homolog of a capsid derived from these, or an artificially modified AAV-derived capsid. As shown in the examples described later, the method of this embodiment makes it possible to encapsulate polynucleotides in the capsid regardless of the serotype from which the capsid originates. This is thought to be because the inclusion of polynucleotides in the capsid proceeds based on a conserved structure common to AAV-derived capsids, and does not depend on sequences specific to a particular serotype.

[0018] As an example, the amino acid sequences of VP1, VP2, and VP3 derived from AAV2 are shown in SEQ ID NOs: 1 to 3. The amino acid sequences of VP1 derived from AAV1, 3 to 13 are shown in SEQ ID NOs: 4 to 15, respectively. That is, VP1 may be a protein containing the amino acid sequence shown in any of SEQ ID NOs: 1, 4 to 15, and preferably a protein containing the amino acid sequence shown in any of SEQ ID NOs: 1, 4, 5, 7, 8, 10, and 11. Furthermore, the amino acid sequences of VP2 from AAV1, 3, 6, 8, and 9 are shown in SEQ ID NOs: 16 to 20, respectively. That is, VP2 may be a protein containing the amino acid sequence shown in any of SEQ ID NOs: 2, 16 to 20. Also, the amino acid sequences of VP3 from AAV1, 3, 6, 8, and 9 are shown in SEQ ID NOs: 21 to 25, respectively. That is, VP3 may be a protein containing the amino acid sequence shown in any of SEQ ID NOs: 3, 21 to 25.

[0019] The capsid proteins may be variants of these, as long as the formed capsid retains the above-mentioned conserved structure and does not significantly impair the titer of the AAV particles. For example, the amino acid sequences shown in SEQ ID NOs: 1 to 15 may include the substitution, deletion, insertion, or addition of one or several amino acids at one or several positions. The term "one or several" above varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein, but specifically means, for example, 1 to 73, 1 to 36, 1 to 10, preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3.

[0020] The above substitutions, deletions, insertions, or additions of one or more amino acids are conservative mutations that maintain the normal function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Glun and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered to be conservative substitutions include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include: e to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu.

[0021] Furthermore, the capsid protein may be a protein having an amino acid sequence that has 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more identity with respect to the entire amino acid sequence, as long as the formed capsid retains the above-mentioned conserved structure and does not significantly impair the titer of the AAV particles. In this specification, "identity" between amino acid sequences means the identity between amino acid sequences calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. Furthermore, "identity" between base sequences means the identity between base sequences calculated using the default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) by blastn.

[0022] Capsid proteins may be chemically modified. The residues that can be chemically modified are not particularly limited, but include lysine residues, cysteine ​​residues, tyrosine residues, asparagine residues, serine residues, threonine residues, the N-terminus, and the C-terminus. Chemical modifications include polyethylene glycolation, drug addition, drug delivery system (DDS) addition, fluorescent labeling, biotinylation, radiolabeling, and glycosylation.

[0023] Examples of drugs that can be added include emtansine, monomethyl auristatin E, monomethyl auristatin F, calicheamicin, doxorubicin, meitansrin, SN-38, pyrilobenzodiazepine, meitansnoid, tansine, and auristatin. These drugs may be added to the capsid protein via a linker. Examples of linkers include hydrazine linkers, disulfide linkers, peptide linkers, non-reducing thioether linkers, and click chemistry linkers.

[0024] The compound added in DDS addition is not particularly limited as long as it is a molecule that controls drug distribution in the body. The method of controlling drug distribution in the body is not particularly limited and includes methods that enhance targeting of target tissues. Examples of molecules that control drug distribution in the body include sugars, polymers, dendrimers, and antibodies. An example of a sugar is N-acetyl-D-galactosamine (GalNaC). An example of a polymer is polyethylene glycol.

[0025] The capsid protein may be a fusion protein with another amino acid sequence. The "other amino acid sequence" can be appropriately selected according to various conditions such as its intended use. Examples of the "other amino acid sequence" include peptide tags, signal sequences, and protease recognition sequences. The "other amino acid sequence" may be linked to or inserted into one or more of the N-terminus, internal, and C-terminus of the capsid protein, for example. The "other amino acid sequence" may be a single amino acid sequence, or a combination of two or more amino acid sequences may be used.

[0026] Peptide tags can be used, for example, for the detection and purification of expressed capsids. Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphate), and the Fc region of antibodies. An example of a His tag is the 6xHis tag.

[0027] Signal sequences can be used, for example, in the secretion and production of capsids. Examples of signal sequences include those recognized by the Sec secretory pathway and those recognized by the Tat secretory pathway. Specifically, examples of signal sequences recognized by the Sec secretory pathway include signal sequences of cell surface proteins of Corynebacteria. Examples of cell surface proteins of Corynebacteria include PS1 (CspA) and PS2 (CspB) of C. glutamicum (Japanese Patent Publication No. 6-502548), and SlpA (CspA) of C. ammoniagenes (C. stationis) (Japanese Patent Publication No. 10-108675). Specifically, examples of signal sequences recognized by the Tat secretory pathway include E. Examples include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013 / 118544). Signal sequences can be used, for example, by being added to the N-terminus of the protein to be produced. Signal sequences are generally cleaved by signal peptidases when the translation product is secreted outside the bacterial cell. Therefore, when secreting capsid proteins using signal sequences, capsid proteins without signal sequences may be secreted outside the bacterial cell.

[0028] The recognition sequence of a protease can be used, for example, to cleave expressed capsid proteins. Preferably, the recognition sequence of the protease is a protease with high substrate specificity. Specifically, examples of protease recognition sequences with high substrate specificity include the recognition sequence of Factor Xa protease, the recognition sequence of proTEV protease, and the recognition sequence of PreScission®Protease. Factor Xa protease recognizes the amino acid sequence of Ile-Glu-Gly-Arg (=IEGR) (SEQ ID NO: 26) in the protein, and proTEV protease recognizes the amino acid sequence of Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO: 27) in the protein, and they specifically cleave the C-terminal side of each sequence. Furthermore, PreScission® Protease recognizes the amino acid sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (=LEVLFQGP) (SEQ ID NO: 28) in proteins and cleaves between Gln and Gly. For example, when expressing a capsid protein as a fusion protein with other amino acid sequences such as peptide tags, by introducing a protease recognition sequence between the capsid protein and the other amino acid sequence, the other amino acid sequence can be removed from the expressed protein using the protease, thereby obtaining a capsid protein that does not contain the other amino acid sequence.

[0029] The capsid is encoded by the cap gene. As an example, the nucleotide sequences of the cap genes for AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, and AAV9 are shown in Sequence IDs 29 to 35, respectively. The nucleotide sequences of the cap genes that can be used in this embodiment are not limited to these sequences, and may include homologous sequences of these cap genes, or sequences that are completely or substantially complementary to these sequences. Furthermore, the cap gene may be a variant in which any codon is replaced with an equivalent codon. That is, the cap gene may be a variant resulting from the degeneracy of the genetic code of the cap gene exemplified above. For example, the cap gene may be modified to have the optimal codon depending on the codon usage frequency of the host being used.

[0030] In this specification, a "sequence that is perfectly complementary" to a given base sequence means a base sequence in which all bases constituting the given base sequence can form Watson-Crick base pairs through hydrogen bonding between the bases at corresponding positions in that sequence. A "sequence that is substantially complementary" to a given base sequence means a base sequence that hybridizes with the given base sequence under stringent conditions. Here, "stringent conditions" refers to conditions in which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, conditions in which highly complementary DNAs, such as a DNA consisting of a certain base sequence, hybridize with DNA having a sequence that is completely complementary to that base sequence, and DNA having a progressively more desirable level of identity, such as 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, and 99% or more, while DNA with lower identity does not hybridize. Alternatively, conditions in which the DNA is washed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, namely 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0031] Gene homologs or protein homologs can be easily obtained from public databases, for example, by BLAST searches or FASTA searches using the nucleotide sequences of the genes or amino acid sequences of the proteins exemplified above as query sequences. Alternatively, gene homologs can be obtained, for example, by PCR using chromosomes from various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of these known genes as primers.

[0032] One method for expressing a capsid in a first cell is to introduce a vector containing the cap gene into the cell. A vector containing the cap gene is also called a cap gene expression vector or a recombinant vector. A cap gene expression vector can be constructed, for example, by ligating a DNA fragment containing the cap gene with a vector that functions in cells. In this specification, "introducing a vector into cells" includes transformation, electroporation, conjugation, infection, transduction, or transfection. As the vector, a vector capable of autonomous replication within the cell can be used. The vector is preferably a multicopy vector. Furthermore, the vector is preferably equipped with an antibiotic resistance gene or the like for drug-assisted selection. The vector may also be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. When constructing an expression vector, for example, the cap gene containing a unique promoter region may be directly incorporated into the vector, the ORF of the cap gene may be bound downstream of the promoter as described above before being incorporated into the vector, or the ORF of the cap gene may be incorporated downstream of the promoter originally present on the vector.

[0033] Vectors, promoters, and terminators usable in various organisms are described in detail in, for example, "Basic Microbiology Course 8: Genetic Engineering," Kyoritsu Shuppan, 1987, and these can be utilized.

[0034] Furthermore, the cap gene can be introduced, for example, onto the chromosomes of cells. Gene introduction onto chromosomes can be carried out, for example, using homologous recombination. Examples of gene introduction methods using homologous recombination include Red-driven integration (WO2005 / 010175), transduction using phages such as P1 phage, methods using conjugation vectors, methods using suicidal vectors that do not have an origin of replication that function in cells, methods using transposons, lentiviral vectors, or retroviral vectors, and methods using genome editing technology. Examples of transposons include piggyback and sleeping beauty. Examples of genome editing technologies include the zinc finger nuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system, or the clustered regularly interspaced short palindromic repeat (CRISPR) / Cas system. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that has multiple copies in the chromosome. Examples of sequences that have multiple copies in the chromosome include repeating DNA sequences and inverted repeats located at both ends of transposons. Furthermore, genes can also be randomly introduced onto chromosomes using methods such as transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, the cap gene containing a unique promoter region may be directly incorporated into the chromosome, the ORF of the cap gene may be bound downstream of the promoter as described above before being incorporated into the chromosome, or the ORF of the cap gene may be incorporated downstream of a promoter that is already present on the chromosome.

[0035] The introduction of a gene onto a chromosome can be confirmed, for example, by Southern hybridization using a probe with a nucleotide sequence complementary to all or part of the gene, or by PCR using primers created based on the nucleotide sequence of the gene.

[0036] Capsid expression can be achieved by culturing cells into which the cap gene described above has been introduced in a culture medium. Gene expression induction may be performed as needed. The culture conditions and gene expression induction conditions for the organism should be appropriately selected according to various conditions such as the type of marker, the type of promoter, and the type of organism. The culture medium used is not particularly limited as long as it allows the organism to grow and express the capsid; any medium known to those skilled in the art can be used.

[0037] In other words, the capsid used in this embodiment may be a protein encoded by a nucleotide sequence that includes the nucleotide sequence shown in any of SEQ ID NOs: 29 to 35, or a nucleotide sequence that is completely or substantially complementary to the nucleotide sequence shown in any of SEQ ID NOs: 29 to 35. Specifically, such protein is VP1, VP2, and / or VP3.

[0038] The manufacturing method of this embodiment may include a step of expressing a capsid in the first cells, followed by a step of disrupting, lysing, or extracting the first cells. Disruption, lysing, or extraction of the first cells can be carried out by known methods. Examples of such methods include sonication, dynomill method, bead disruption, French press disruption, lysozyme treatment, and dissolution using compounds such as surfactants. These methods may be used individually, or two or more may be used in appropriate combinations. Furthermore, for example, if the capsid accumulates in the culture medium, the culture supernatant can be obtained by centrifugation or the like, and the capsid can be purified from the culture supernatant.

[0039] In this embodiment, the manufacturing method includes, in one example, a step of synthesizing a capsid using a cell-free synthesis system. Here, a cell-free synthesis system refers to a method of synthesizing mRNA and proteins encoded by a nucleic acid (DNA or mRNA) in vitro from a template nucleic acid, using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering methods), rather than using living cells. In a cell-free synthesis system, a cell extract obtained by purifying a cell lysate as needed is generally used. The cell extract generally contains ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, which are necessary for protein synthesis. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to this cell extract. During protein synthesis, separately prepared ribosomes, various factors, and / or various enzymes may be supplemented as needed.

[0040] The capsid used in the manufacturing method of this embodiment may be purified. Methods for purifying the capsid include, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, size exclusion chromatography, isoelectric focusing precipitation, centrifugation, ultracentrifugation, dialysis, filter separation, reverse-phase chromatography, and ion-pair chromatography. These methods may be used individually or in combination of two or more as appropriate. The capsid can be purified to a desired degree. The purity of the purified capsid may be, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% or higher. The purity of the capsid can be measured by SDS-PAGE or liquid-phase chromatography. When using commercially available capsids, catalog values ​​may be used.

[0041] <Rep Protein> The Rep protein used in this embodiment is synthesized in a second system. The second system may use a second cell that expresses the Rep protein. That is, the production method of this embodiment may include a step of expressing the Rep protein in the second cell. Alternatively, the second system may utilize a cell-free synthesis system. That is, the production method of this embodiment may include a step of synthesizing the Rep protein using a cell-free synthesis system.

[0042] The second cell used in the process of expressing the Rep protein in the second cell is not particularly limited as long as it is capable of expressing the Rep protein. Examples of cells include bacteria, actinomycetes, yeasts, fungi, plant cells, insect cells, and mammalian cells. Examples of bacteria include coryneform bacteria such as Escherichia coli and Corynebacterium glutamicum. Examples of yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastris. Examples of fungi include filamentous fungi. Examples of plant cells include tobacco. Examples of insect cells include Sf9 cells. Examples of mammalian cells include human cells such as HEK293 cells, CHO cells, and Hela cells. Among these, Escherichia coli, coryneform bacteria, filamentous fungi, HEK293 cells, Saccharomyces cerevisiae, CHO cells, Sf9 cells, tobacco, Schizosaccharomyces pombe, or Pichia pastris are preferred, with Escherichia coli or coryneform bacteria being more preferred.

[0043] The second cell may be of the same species as the first cell, or it may be of a different species.

[0044] The Rep protein is preferably one or more selected from Rep78, Rep68, Rep52, and Rep40, and more preferably one or more selected from Rep78 and Rep68. The Rep protein may also be two or more selected from Rep78, Rep68, Rep52, and Rep40. The Rep protein preferably contains Rep78 or Rep68 and Rep52 or Rep40. If the Rep protein is two or more selected from Rep78, Rep68, Rep52, and Rep40, the two or more Rep proteins may be expressed in different cells or in the same cell.

[0045] Rep78, Rep68, Rep52, and Rep40 are isoforms encoded by the rep gene, but differ in their transcription start site and / or splicing. Rep78 and Rep68 have a domain involved in nicking activity at the N-terminus and a domain responsible for ATPase activity and DNA helicase activity at the C-terminus. On the other hand, Rep52 and Rep40 have domains responsible for ATPase activity and DNA helicase activity, but lack the domain involved in nicking activity at the N-terminus. The inventors have found that ATPase activity and / or DNA helicase activity are important for the function of encapsulating a polynucleotide encoding a target sequence in a capsid, while nicking activity, which introduces single-strand breaks (nicks) into ATPase-active double-stranded DNA, is not essential. In other words, as long as it has ATPase activity and / or DNA helicase activity, one or more selected from Rep78, Rep68, Rep52, and Rep40 can be used to encapsulate a polynucleotide encoding the target sequence in a capsid.

[0046] The Rep protein may be derived from AAV, specifically AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, or AAVrh10, with AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV12, or AAV13 being preferred. It may also be a homolog or mutant of the Rep protein derived from these. Examples of mutants include mutant Rep proteins having mutations such as substitution, deletion, or insertion of amino acid residues in the amino acid sequence, and chimeric Rep proteins combining amino acid sequences from different serotypes. As shown in the examples described later, the method of this embodiment makes it possible to encapsulate polynucleotides in the capsid regardless of the serotype from which the Rep protein originates. This is thought to be because the encapsulation of polynucleotides in the capsid proceeds based on a conserved structure common to AAV-derived Rep proteins and does not depend on sequences specific to a particular serotype.

[0047] The origin of the Rep protein may be the same as or different from the origin of the capsid. That is, any serotype of Rep protein can be combined with any serotype of capsid; for example, a combination of AAV2-derived Rep protein and AAV8-derived capsid may be used.

[0048] As an example, the amino acid sequences of Rep78, Rep68, Rep52, and Rep40 derived from AAV2 are shown in SEQ ID NOs: 36-39. In addition, the amino acid sequences of Rep78 derived from AAV1, 3-13 are shown in SEQ ID NOs: 40-51, respectively.

[0049] Rep proteins may be any of these mutants, as long as they have the function of encapsulating a polynucleotide encoding the target sequence in the capsid. In particular, the inventors have found that ATPase activity and / or DNA helicase activity are important for the function of encapsulating a polynucleotide encoding the target sequence in the capsid, while nicking activity, which introduces single-strand breaks (nicks) into double-stranded DNA, is not essential. In other words, having the function of encapsulating a polynucleotide encoding the target sequence in the capsid can be rephrased as having ATPase activity and / or DNA helicase activity. Furthermore, it is known that a mutant of the AAV2-derived Rep68 protein in which the tyrosine at the 156th residue is replaced with phenylalanine (Y156F mutant, SEQ ID NO: 52) shows a significant decrease or inactivation of nicking activity. Therefore, Rep proteins may be mutants of the Y156F mutant with further mutations, as long as they have ATPase activity and / or DNA helicase activity.

[0050] The Rep protein may be a protein containing an amino acid sequence that includes the substitution, deletion, insertion, or addition of one or several amino acids at one or several positions in the amino acid sequence shown in any of SEQ ID NOs: 36 to 52, preferably in any of SEQ ID NOs: 36, 37, 41 to 44, 46, 47, or 50 to 52. The above "one or several" will vary depending on the position and type of amino acid residues in the three-dimensional structure of the protein, but specifically it means, for example, 1 to 53, 1 to 26, 1 to 10, preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3.

[0051] The substitutions, deletions, insertions, or additions of one or more amino acids described above are conservative mutations that maintain the normal function of the protein. The above explanation applies to conservative mutations.

[0052] Furthermore, the Rep protein may be a protein having an amino acid sequence that is 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more identical to the entire amino acid sequence, as long as it has the function of encapsulating a polynucleotide encoding the target sequence in a capsid.

[0053] The Rep protein may be any of the above-mentioned proteins, excluding those whose amino acid sequence or the base sequence of the gene encoding it is publicly known at the time of filing the application of the present invention.

[0054] The Rep protein may also be a fusion protein with other amino acid sequences. The above explanation applies to other amino acid sequences. Examples of fusion proteins with other amino acid sequences include fusion proteins with tag sequences. Here, the tag sequence may be, for example, a soluble protein (also called a solubilizing tag). By fusing a soluble protein, the expression of the Rep protein in cells expressing it can be improved. Examples of soluble proteins include GFP protein (green fluorescent protein), GST protein (glutathione S-transferase protein), HA protein (influenza hemagglutinin protein), MBP protein (maltose-binding protein), Thioredoxin protein, and SUMO protein (small ubiquitin-like modified protein).

[0055] The Rep protein is encoded by the rep gene. As an example, the nucleotide sequence of the AAV2 rep gene is shown in Sequence ID No. 53. The nucleotide sequence of the rep gene that can be used in this embodiment is not limited to this sequence, and may include the homolog sequence of the AAV2 rep gene, or a sequence that is completely or substantially complementary to these sequences. Furthermore, the rep gene may be a variant in which any codon is replaced with an equivalent codon. That is, the rep gene may be a variant resulting from the degeneracy of the genetic code of the rep gene exemplified above. For example, the rep gene may be modified to have the optimal codon depending on the codon usage frequency of the host being used.

[0056] One method for expressing the Rep protein in a second cell is to introduce a vector containing the rep gene into the cell. A vector containing the rep gene is also called a rep gene expression vector or recombinant vector. A rep gene expression vector can be constructed, for example, by ligating a DNA fragment containing the rep gene with a vector that functions in cells. Examples of such vectors include those similar to those containing the cap gene.

[0057] Furthermore, the rep gene can be introduced, for example, onto the chromosomes of a cell. The above explanation will be used to describe the introduction of genes onto chromosomes.

[0058] The manufacturing method of this embodiment may include a step of disrupting, lysing, or extracting the second cells after the step of expressing the Rep protein in the second cells. Disrupting, lysing, or extracting the second cells can be done in the same way as disrupting, lysing, or extracting the first cells. Furthermore, for example, if the Rep protein accumulates in the culture medium, the culture supernatant can be obtained by centrifugation or the like, and the Rep protein can be purified from the culture supernatant.

[0059] In one example, the manufacturing method of this embodiment includes a step of synthesizing Rep protein using a cell-free synthesis system. The cell-free synthesis system is described in the above explanation.

[0060] The Rep protein used in the manufacturing method of this embodiment may be purified. Examples of methods for purifying the Rep protein include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, size exclusion chromatography, isoelectric point precipitation, centrifugation, ultracentrifugation, dialysis, filter separation, reverse-phase chromatography, and ion pair chromatography. These methods may be used individually or in combination of two or more as appropriate. The Rep protein can be purified to a desired degree. The purity of the purified Rep protein may be, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% or higher. If the Rep protein is a fusion protein with a soluble protein, the purity of the purified Rep protein is preferably 30% or higher. If the Rep protein is not a fusion protein with another amino acid sequence, the purity of the purified Rep protein is preferably 80% or higher. The purity of Rep protein can be measured by SDS-PAGE or liquid-phase chromatography. If commercially available Rep protein is used, the catalog value may be used.

[0061] <Step of encapsulating the polynucleotide in the capsid> The manufacturing method of this embodiment includes the step of mixing the capsid, the Rep protein, and the polynucleotide encoding the target sequence, and encapsulating the polynucleotide in the capsid. This produces recombinant adeno-associated virus (rAAV) particles in which the polynucleotide encoding the target sequence is encapsulated in the capsid. This step may be carried out as an in vitro system or as an extracellular reaction. The manufacturing method of this embodiment may be a system that does not occur naturally, or a system that does not occur in the human body or animal body.

[0062] The polynucleotide encoding the target sequence may be DNA, RNA, or a combination thereof. Furthermore, the polynucleotide encoding the target sequence may be single-stranded or double-stranded, but preferably double-stranded. That is, the polynucleotide encoding the target sequence may be single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. Examples of single-stranded polynucleotides include mRNA and antisense oligonucleotides. Examples of double-stranded polynucleotides include siRNA. The polynucleotide encoding the target sequence may also contain both DNA residues and RNA residues within a single polynucleotide chain. When the polynucleotide contains RNA, descriptions of DNA, such as the base sequence, may be appropriately interpreted to reflect RNA. The polynucleotide encoding the target sequence may be circular or linear. As shown in the examples described later, the polynucleotide encoding the target sequence is preferably linear in order to allow for the preparation of AAV particles containing the target sequence with high purity. In conventional technology, cyclic polynucleotides are used exclusively, and the selectivity of the encapsulated sequence is not adequately considered. Furthermore, the polynucleotide encoding the target sequence may have a hairpin structure. If the polynucleotide encoding the target sequence has a hairpin structure, it may have hairpin structures at both ends, at one end, or internally. The form of the polynucleotide can be appropriately selected according to various conditions such as its intended use. The polynucleotide encoding the target sequence may be an exogenous polynucleotide. Furthermore, the polynucleotide encoding the target sequence may be a polynucleotide of a different origin than the capsid or Rep protein.

[0063] Linear double-stranded DNA can be prepared, for example, by cleaving a plasmid with restriction enzymes, amplifying a target fragment by PCR, cleaving PCR products with restriction enzymes, amplifying a target fragment by Rolling Circle Amplification and then cleaving it with restriction enzymes, or by chemical synthesis. Linear single-stranded DNA can be prepared, for example, by asymmetric PCR, enzymatically degrading one strand of linear double-stranded DNA, denaturing and separating linear double-stranded DNA, synthesizing DNA using reverse transcriptase with RNA as a template and then digesting the RNA, annealing linear single-stranded DNA, or by chemical synthesis. Linear single-stranded RNA can be prepared, for example, by in vitro transcription, extraction from cells, or by chemical synthesis. Linear double-stranded RNA can be prepared, for example, by annealing linear single-stranded RNA, or by synthesis using RNA-dependent RNA polymerase.

[0064] The polynucleotide used in the manufacturing method of this embodiment may be purified. Methods for purifying the polynucleotide include, for example, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, size exclusion chromatography, ethanol precipitation, isopropanol precipitation, PEG precipitation, phenol / chloroform extraction, TRIZOL / AGPC method, centrifugation, ultracentrifugation, dialysis, filter separation, reversed-phase chromatography, and ion-pair chromatography. These methods may be used individually or in combination of two or more as appropriate. The purification of the polynucleotide can be performed to a desired degree. The purity of the purified polynucleotide may be, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% or higher. The purity of the polynucleotide can be measured by ultraviolet absorption spectroscopy or liquid-phase chromatography. When using commercially available polynucleotides, catalog values ​​may be used.

[0065] The length of the polynucleotide encoding the target sequence is not particularly limited, but is preferably 20 to 5,000 bases, more preferably 30 to 5,000 bases, even more preferably 50 to 5,000 bases, and even more preferably 130 to 4,850 bases. Furthermore, when encapsulating oligonucleotides such as antisense oligonucleotides, the length may be 10 to 300 bases, preferably 15 to 150 bases, and more preferably 18 to 50 bases.

[0066] The target sequence is not particularly limited; it may or may not encode a protein.

[0067] The polynucleotide encoding the target sequence may or may not contain part or all of the ITR. "ITR" refers to a terminal inverted repeat sequence derived from the AAV genome, consisting of approximately 145 bases, and is a sequence structure that can form a T-shaped hairpin structure. Conventionally, it was thought that at least part of the ITR was necessary at both ends of the polynucleotide for the production of a capsid containing a polynucleotide encoding the target sequence, but according to the manufacturing method of this embodiment, the polynucleotide can be encapsulated in the capsid even if part or all of the ITR is not present at both ends of the polynucleotide. The ITR may be derived from AAV, and may be derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, AAVDJ8, or AAVrh10, AAVrh74, or AAVhu37. As an example, the nucleotide sequence of the ITR derived from the 5' end of AAV2 is shown in SEQ ID NO: 54. In this specification, the ITR is not limited to the above sequence as long as it enables viral replication, and may include, for example, a sequence that is completely or substantially complementary to the nucleotide sequence shown in SEQ ID NO: 54. That is, including the entire ITR may include the nucleotide sequence shown in SEQ ID NO: 54, or it may include the nucleotide sequence shown in SEQ ID NO: 54 or a sequence that is completely or substantially complementary to the nucleotide sequence shown in SEQ ID NO: 54. Not including the entire ITR may not include the nucleotide sequence shown in SEQ ID NO: 54, or it may not include a sequence that includes the nucleotide sequence shown in SEQ ID NO: 54 and a sequence that is completely or substantially complementary to the nucleotide sequence shown in SEQ ID NO: 54. Furthermore, as part of the ITR, for example, positions 9-42, 18-33, 43-49, 55-61, 43-61, 65-71, 77-83, 65-83, 84-125, 93-108, or 126-145 of the nucleotide sequence shown in SEQ ID NO: 54, or sequences that are completely or substantially complementary to these nucleotide sequences. Positions 43-61 and 65-83 of the nucleotide sequence shown in SEQ ID NO: 54 are known as the B-B' region and C-C' region, respectively, and are known not to be essential for polynucleotide inclusion (Zhou et al., Sci. Rep., 7(1): 5432 (2017)).Furthermore, the nucleotide sequences at positions 93-108 of the nucleotide sequence shown in Sequence ID No. 54 and its complementary sequence at positions 18-33 are called Rep binding elements (RBEs), and are known as important cis-elements for the function of ITR because they are sequences recognized and bound by Rep78 / Rep68. However, as described above, according to the manufacturing method of this embodiment, polynucleotides can be encapsulated in the capsid even when polynucleotides that do not contain RBEs are used. In another embodiment, a portion of the ITR may be a sequence that includes the nucleotide sequence shown in Sequence ID No. 55 or a sequence that is completely or substantially complementary to the nucleotide sequence shown in Sequence ID No. 55. In other words, including a part of the ITR may include a sequence selected from the group consisting of the nucleotide sequences shown in Sequence ID No. 54, positions 9-42, 18-33, 43-49, 55-61, 43-61, 65-71, 77-83, 65-83, 84-125, 93-108, and 126-145, as well as the nucleotide sequence shown in Sequence ID No. 55, and sequences that are completely or substantially complementary to these nucleotide sequences. The absence of a portion of the ITR may mean that the sequence does not include positions 9-42, 18-33, 43-49, 55-61, 43-61, 65-71, 77-83, 65-83, 84-125, 93-108, and 126-145 of the sequence shown in Sequence ID No. 54, nor the sequence shown in Sequence ID No. 55, nor sequences that include sequences that are completely or substantially complementary to these sequences. The absence of positions 9-42, 18-33, 84-125, 93-108, and 126-145 of the sequence shown in Sequence ID No. 54, nor the sequence shown in Sequence ID No. 55, nor the absence of positions 18-33 and 93-108 of the sequence shown in Sequence ID No. 54.

[0068] If the polynucleotide encoding the target sequence contains part or all of the ITR, part or all of the ITR may be located at the 5' end, the 3' end, or both ends of the target sequence. If the polynucleotide encoding the target sequence is linear, it is preferable that part or all of the ITR be located at the end.

[0069] The polynucleotide encoding the target sequence may have modified residues. Modified residues include substitution of substituents on the sugar portion (ribose or deoxyribose) of the nucleotide residue, substitution of the sugar portion (sugar backbone) of the nucleotide residue, substitution of the base portion of the nucleotide residue, or substitution of the phosphate diester portion of the nucleotide residue.

[0070] In the manufacturing method of this embodiment, when a polynucleotide containing a modified residue is used, the rAAV particles produced contain a polynucleotide containing a modified residue at a predetermined position. The predetermined position may be at the end of the polynucleotide or in the interior. Examples of the predetermined position include one or more locations between 1 and 50 bases from the 5' end of the polynucleotide, or one or more locations between 1 and 25 bases. Examples of the predetermined position include one or more locations between 1 and 50 bases from the 3' end of the polynucleotide, or one or more locations between 1 and 25 bases. Furthermore, the predetermined position may, for example, be at the 5' end or the 3' end of the polynucleotide. Furthermore, the predetermined position may be some or all of a specific type of base contained in the polynucleotide.

[0071] The proportion of polynucleotides containing a modified residue at the predetermined position among all polynucleotides contained in rAAV particles is not particularly limited, but may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. The proportion of polynucleotides containing a modified residue at the predetermined position among all polynucleotides contained in rAAV particles can be measured according to the type of modified residue, for example, by mass spectrometry.

[0072] Examples of "substitution of substituents on the sugar portion of nucleotide residues" include substitution of the hydrogen or hydroxyl group at the 1', 2', 3', 4', or 5' position of ribose or deoxyribose with other substituents. Substitution of substituents on the sugar portion of nucleotide residues can improve the degradation resistance of polynucleotides. Furthermore, if the other substituent is a ligand, it can improve or add properties such as stability, targeting, and pharmacokinetics. It is preferable that the polynucleotide encoding the target sequence has substitution of the hydrogen or hydroxyl group at the 2' position of ribose or deoxyribose with other substituents.

[0073] Other substituents to which the hydrogen or hydroxyl group at the 1', 2', 3', 4', or 5' position may be substituted include one or more selected from the group consisting of halogen atoms, alkyl groups, oxyalkyl groups, aryl groups, heteroaryl groups, nitrogen-containing non-aromatic heterocyclic groups, amino groups, azide groups, and ligands. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. Preferred alkyl groups are C1-C6 alkyl groups (hereinafter sometimes referred to as "C1-6 alkyl"). In addition, one or more hydrogens of the alkyl group may be substituted by other substituents. Examples of substituents on the alkyl group include one or more selected from the group consisting of halogen atoms, amino groups, C1-C6 alkoxy groups, C1-C6 alkoxycarbonyl groups, C1-C6 monoalkylcarbonyl groups, and C1-C6 dialkylcarbonyl groups. Here, "C1-C6" refers to the number of carbon atoms in the alkoxy group or alkyl group included in these substituents. The alkoxycarbonyl group, monoalkylcarbonyl group, and dialkylcarbonyl group may be further substituted with one or more groups selected from the group consisting of aryl groups having 6 to 14 carbon atoms, heteroaryl groups having 5 to 10 members, and nitrogen-containing non-aromatic heterocyclic groups having 3 to 11 members. The alkyl group included in the oxyalkyl group is the same as that of the alkyl group described above. Examples of aryl groups include aryl groups having 6 to 14 carbon atoms (hereinafter sometimes referred to as "C6-14 aryl"). Examples of heteroaryl groups include heteroaryl groups having 5 to 10 members. Examples of nitrogen-containing non-aromatic heterocyclic groups include nitrogen-containing non-aromatic heterocyclic groups having 3 to 11 members. One or two hydrogen atoms of the amino group may be substituted with other substituents. Substituents that can be attached to the amino group include pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, 4-tert-butylphenoxyacetyl group, acetyl group, benzoyl group, isobutyryl group, dimethylformamidinyl group, and 9-fluorenylmethyloxycarbonyl group. Other substituents that can be substituted with a hydrogen or hydroxyl group at the 1', 2', 3', 4', or 5' position include dimethylaminoethoxyethyl group.

[0074] More specifically, other substituents that may be substituted with hydrogen or hydroxyl groups at the 1', 2', 3', 4', or 5' positions include, for example, C1-6 alkyloxy C1-6 alkyl (e.g., methoxyethyl: MOE), -O-C1-6 alkyl (e.g., -O-Me), -O-C6-14 aryl (e.g., -O-phenyl), -C-aryl (e.g., -C-phenyl), halogen atoms (e.g., fluorine atoms), -O-C1-6 alkylN-amide C1-6 alkyl (e.g., -O-N-methylacetamide, -O-NMA), -O-C1-6 alkyl-(C1-6 alkyl-)amino-C1-6 alkyloxy C1-6 alkyl (e.g., -O-dimethylaminoethoxyethyl, -O-DMAEOE), and -O-amino C1-6 alkyl (e.g., -O-aminopropyl, -O-AP).

[0075] Among the above, it is preferable to have substitution of the hydroxyl group at the 2' position, for example, 2'-C1-6 alkyloxy C1-6 alkyl (e.g., 2'-methoxyethyl), 2'-O-C1-6 alkyl (e.g., 2'-O-Me), 2'-O-C6-14 aryl (e.g., 2'-O-phenyl), 2'-C-aryl (e.g., 2'-C-phenyl), 2'-halogen atom (e.g., 2'-F), 2'-O-C1-6 alkylN-amide C1-6 alkyl (e.g., 2'-O-N-methylacetamide, 2'-O-NMA), 2'-O-C1-6 alkyl-(C1-6 alkyl-)amino-C1-6 alkyloxy C1-6 alkyl (e.g., 2'-O-dimethylaminoethoxyethyl, 2'-O-DMAEOE), and 2'-O-amino C1-6 alkyl (e.g., 2'-O-aminopropyl, 2'-O-AP). Among these, it is more preferable to have one or more substituents selected from the group consisting of 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-phenyl, and 2'-O-dimethylaminoethoxyethyl.

[0076] Furthermore, if a hydrogen or hydroxyl group at the 1', 2', 3', 4', or 5' position is substituted, these two substitution sites may be crosslinked. Examples of crosslinked positions include the hydroxyl group at the 2' position and the hydrogen at the 4' position, and the hydrogen at the 3' position and the hydrogen at the 5' position. Examples of structures in which the hydroxyl group at the 2' position and the hydrogen at the 4' position, or the hydrogen at the 3' position and the hydrogen at the 5' position, include structures in which the 2' and 4' positions, or the 3' and 5' positions, are crosslinked by an alkylene group having 2 to 6 carbon atoms. Here, one or more hydrogens of the alkylene group having 2 to 6 carbon atoms may be substituted by an alkyl group having 1 to 6 carbon atoms. Furthermore, one or two methylene groups of a C2-C6 alkylene group may be substituted with -O-, -NR-, -S-, -CO-, -CS-, -COO-, -OCONR-, -CONR-, or -CSNR- (where R represents a hydrogen atom or a C1-C6 alkyl group). Specifically, 2'-O-methylene-4' (also called "Loc nucleic acid" or "LNA"), 2'-O-ethylene-4' (also called "ethylene-crosslinked nucleic acid" or "ENA"), 2'-O-methyl-substituted methylene-4' (also called "constrand-ethyl crosslinked nucleic acid" or "cEt"), and 2'-O-methylene-O-methylene-4' ("BNA"). ＣＯＣ Also known as "BNA"), 2'-O-N(R)-methylene-4' (R represents a methyl group, a hydrogen atom, or a benzyl group). ＮＣ Examples include 2'-N(methyl)-C(O)-4' (also known as "amide-bridged nucleic acid" or "AmNA"), 2'-NH-methylene-4', 2'-methyl-substituted ethylene-4', and 3'-ethylene-5' (also known as "bicyclonucleotide" or "Bc nucleic acid").

[0077] Ligands include N-acetylgalactosamine, peptides, phosphates, cholesterol, cholic acid, tocopherol, adamantane acetate, palmitic acid, myristic acid, polyethylene glycol, folic acid, mannose-6-phosphate, galactose, fructose, ribose, xylose, arabinose, lyxose, allose, altrose, growth, iodose, glucose, and talose.

[0078] Modified residues involving "substitution of the sugar portion of the nucleotide residue itself" include, for example, nucleotide residues in oligonucleotides such as hexitol nucleic acid (HNA) and cyclohexenyl nucleic acid (CeNA) that involve substitution of a five-membered ring sugar with a six-membered ring pseudosugar. Furthermore, modified residues involving "substitution of the sugar portion of the nucleotide residue itself" also include morpholino nucleic acid (PMO) residues, which are nucleotide-like artificial compounds having a morpholino ring structure that is not degraded by enzymes in the body (e.g., nucleases such as RNase) and does not induce an immune response.

[0079] Examples of "substitutions of the base portion of nucleotide residues" include the substitution of a methyl group from the hydrogen at position 5 of cytosine (a base having this substitution is also called "5-methylcytosine" or "5mC"), the substitution of a carbon at position 7 of guanine (a base having this substitution is also called "7-deazaguanine"), the substitution of a nitrogen at position 5 of cytosine (a base having this substitution is also called "5-azacytosine"), and the substitution of a sulfur at position 4 of uracil (a base having this substitution is also called "4-thiouracil").

[0080] Examples of "substitution of the phosphate diester portion of a nucleotide residue" include substitution of a non-crosslinked oxygen atom to a sulfur atom in the phosphate diester portion, substitution of a crosslinked oxygen atom to a secondary amino group in the phosphate diester portion, and substitution of a hydroxyl group to OR' in the phosphate diester portion. Here, R' represents an alkyl group having 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms, or a 5 to 10-membered heteroaryl group. One or more hydrogen atoms of the alkyl group having 1 to 6 carbon atoms may be substituted with a halogen atom, a cyano group, an aryl group having 6 to 10 carbon atoms, or a 5 to 10-membered heteroaryl group. One or more hydrogen atoms of the cycloalkyl group having 3 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms, and a 5 to 10-membered heteroaryl group may be substituted with an alkyl group having 1 to 6 carbon atoms, a halogen atom, or a cyano group.

[0081] The method for producing polynucleotides containing modified residues is not particularly limited and includes chemical synthesis methods and enzyme-based methods. A chemical synthesis method, for example, can be produced by a method comprising: a first step of preparing a nucleoside or polynucleotide having protecting groups at the elongation site and non-elongation site; a second step of removing the protecting group at the elongation site; a third step of reacting a nucleoside having a hydroxyl group with the elongation site and condensing them via a phosphite diester bond; a fourth step of converting the phosphite diester bond to a phosphate diester bond, a thiophosphate diester bond, an aminophosphate diester bond, a boranophosphate diester bond, a phosphate diester bond protected by a protecting group (phosphate triester bond), or a thiophosphate diester bond protected by a protecting group (thiophosphate-O,O,S-tryster bond), etc.; and a fifth step of removing the protecting group. Here, the nucleoside having a hydroxyl group may be the modified residue. Furthermore, the nucleoside having a hydroxyl group may have a protecting group. The polynucleotide obtained by converting the aforementioned phosphite diester bond to a phosphate diester bond or the like may be used as the polynucleotide in the first step. By repeating the first to fourth steps in this manner, a polynucleotide with a desired number of bases and modified residues at predetermined positions can be produced.

[0082] Enzyme-based methods include performing PCR in a reaction solution containing modified primers and / or nucleotide triphosphates (NTPs), and modifying polynucleotides by reacting them with enzymes. Modified primers can be synthesized, for example, by the chemical synthesis method described above. When modified primers are used, the produced polynucleotides contain modified residues at the predetermined positions of the primers. When modified NTPs are used, the type and proportion of modified residues in the produced polynucleotides can be controlled by adjusting the type and proportion of modified NTPs among the NTPs used in the PCR reaction.

[0083] Furthermore, when performing PCR, a primer containing a modified residue at a predetermined position may be used in combination with an NTP having the above modification.

[0084] Enzymes that act on polynucleotides include DNA methyltransferase, DNA hydroxymethyltransferase, RNA methyltransferase, polynucleotide kinase, phosphatase, alkaline phosphatase, DNA glycosylase, and base editing enzymes. Alternatively, a ligase may be used as the enzyme to ligate polynucleotides with oligonucleotides containing modifying groups.

[0085] As described above, the polynucleotides used in this embodiment do not need to be designed to be replicated within cells. Therefore, the polynucleotides used in this embodiment are not limited to DNA replicated within cells. In other words, polynucleotides that could not be encapsulated in the capsid using conventional methods for producing rAAV particles can be encapsulated in the capsid. That is, another embodiment of the present invention is a recombinant adeno-associated virus particle in which a) a modified residue is contained, b) some or all of the ITR is not contained, or c) a polynucleotide containing RNA is encapsulated in the capsid.

[0086] Water is a suitable solvent for mixing the capsid and the Rep protein with the polynucleotide encoding the target sequence. That is, this step prepares a composition comprising the capsid, the Rep protein, and the polynucleotide encoding the target sequence, which is preferably an aqueous solution. Another embodiment of the present invention is a composition comprising the capsid, the Rep protein, and a polynucleotide that a) contains modified residues, b) does not contain some or all of the ITR, or c) contains RNA.

[0087] The concentration of the capsid in the composition is sufficient to encapsulate the polynucleotide within the capsid. The capsid concentration is preferably 1 pM or higher, more preferably 10 pM or higher, even more preferably 100 pM or higher, and particularly preferably 1 nM or higher. There is no upper limit, but examples include 1 μM or less, 100 nM or less, 50 nM or less, and 20 nM or less. That is, the capsid concentration in the composition may be 1 pM to 1 μM, 10 pM to 100 nM, 100 pM to 50 nM, or 1 nM to 20 nM. In other words, in the encapsulation step, the capsid may be mixed so that the capsid concentration falls within the above range.

[0088] The concentration of the Rep protein in the composition is sufficient to encapsulate the polynucleotide in the capsid. The concentration of the Rep protein is preferably 1 pM or higher, more preferably 10 pM or higher, even more preferably 100 pM or higher, even more preferably 1 nM or higher, and particularly preferably 10 nM or higher. There is no upper limit, but examples include 100 μM or less, 10 μM or less, 1 μM or less, and 100 nM or less. That is, the concentration of the Rep protein in the composition may be 1 pM to 100 μM, 10 pM to 10 μM, 100 pM to 10 μM, 1 nM to 10 μM, 1 nM to 1 μM, 10 nM to 1 μM, or 10 nM to 100 nM. That is, in the encapsulation step, the Rep protein may be mixed so that the concentration of the Rep protein falls within the above range.

[0089] The concentration of the polynucleotide encoding the target sequence in the composition is sufficient to encapsulate the polynucleotide in the capsid. The concentration of the polynucleotide is preferably 1 pM or higher, more preferably 10 pM or higher, even more preferably 100 pM or higher, and particularly preferably 1 nM or higher. There is no upper limit, but examples include 1 μM or less, 100 nM or less, 50 nM or less, and 20 nM or less. That is, the concentration of the polynucleotide in the composition may be 1 pM to 1 μM, 10 pM to 100 nM, 100 pM to 50 nM, or 1 to 20 nM. In other words, in the encapsulation step, the polynucleotide may be mixed so that the concentration of the polynucleotide falls within the above range.

[0090] The amount of the polynucleotide relative to the capsid is not particularly limited, but is preferably 0.1 to 100 in molar ratio, and more preferably 0.1 to 2.

[0091] The composition may contain a buffering agent. Examples of buffering agents include phosphates, citrates, carbonates, acetates, borates, trishydroxymethylaminomethane, GOOD buffers (e.g., HEPES-NaOH buffers), glycine, glycylglycine, and imidazole.

[0092] The composition may contain ATP. The concentration of ATP is not particularly limited, but may be 0.01 mM or more, 0.1 mM or more, 0.3 mM or more, or 1 mM or more. It may also be 500 mM or less, 100 mM or less, 40 mM or less, or 10 mM or less. In other words, the concentration of ATP in the composition may be 0.01 to 500 mM, 0.1 to 100 mM, 0.3 to 40 mM, or 1 to 10 mM.

[0093] The above composition may contain divalent metal salts. Examples of divalent metal salts include magnesium salts, zinc salts, manganese salts, calcium salts, iron salts, and cobalt salts. Among these, magnesium salts are preferred. Examples of counterions to divalent metal ions include sulfate ions, chloride ions, and acetate ions. Specifically, examples of divalent metal salts include magnesium sulfate, magnesium chloride, magnesium acetate, zinc sulfate, zinc chloride, zinc acetate, manganese sulfate, manganese chloride, manganese acetate, calcium sulfate, calcium chloride, calcium acetate, iron sulfate, iron chloride, iron acetate, cobalt sulfate, cobalt chloride, and cobalt acetate.

[0094] The concentration of the divalent metal salt is not particularly limited, but may be 0.1 mM or higher, or 1 mM or higher. It may also be 100 mM or lower, or 30 mM or lower. In other words, the concentration of the divalent metal salt in the composition may be 0.1 to 100 mM, or 1 to 30 mM.

[0095] The above composition may or may not contain commonly used additives such as salts other than divalent metal salts, pH adjusters, antioxidants, thickeners, stabilizers, and emulsifiers.

[0096] Other than divalent metal salts, monovalent cation salts can be mentioned. Examples of monovalent cation salts include salts of monovalent cations such as lithium ions, sodium ions, potassium ions, rubidium ions, cesium ions, and ammonium ions with anions such as fluoride ions, chloride ions, bromide ions, and iodide ions. These monovalent cation salts may be used individually, or two or more may be used in appropriate combinations.

[0097] The concentration of the monovalent cation salt is preferably 1 mM or higher, more preferably 50 mM or higher, and particularly preferably 100 mM or higher. It is also preferably 500 mM or lower, more preferably 400 mM or lower, and even more preferably 300 mM or lower. In other words, the concentration of the monovalent cation salt in the composition may be 1 to 500 mM, 50 to 400 mM, or 100 to 300 mM. Furthermore, if the composition contains two or more types of monovalent cation salts, their total concentration may be within the above range. If the concentration of the monovalent cation salt is below the above upper limit, it is less likely to inhibit the polymerization of Rep proteins or the complex formation with capsids and polynucleotides. Furthermore, if the concentration of the monovalent cation salt is above the above lower limit, the stability of the capsid tends to improve.

[0098] Antioxidants include reducing agents such as dithiothreitol, mercaptoethanol, and tris(2-carboxyethyl)phosphine. Stabilizers include bovine serum albumin (hereinafter sometimes referred to as "BSA"), polyethylene glycol (hereinafter sometimes referred to as "PEG"), and glycerol.

[0099] The composition may contain a nonionic water-soluble polymer. A polymer being water-soluble means that it dissolves in water at concentrations described later, for example, and forms a fluid solution. Preferably, the water-soluble polymer is a chemically substantially inert polymer. In this specification, the nonionic water-soluble polymer may be any surfactant other than those described later. The inclusion of a nonionic water-soluble polymer in the composition may increase the encapsulation efficiency of polynucleotides into the capsid. The mechanism by which encapsulation efficiency increases is not particularly limited, but it is thought that the presence of a water-soluble polymer at a high concentration in the composition causes a phenomenon called molecular crowding, which reduces the effective reaction field and increases the substantial local concentration of the capsid, Rep protein, and polynucleotide, thereby creating an environment that facilitates encapsulation of polynucleotides into the capsid. Examples of the water-soluble polymer include, but are not limited to, polyether-based water-soluble polymers such as polyethylene glycol (PEG) and poly(propylene glycol); water-soluble polysaccharides such as dextran and cellulose derivatives; vinyl-based water-soluble polymers such as polyvinylpyrrolidone; and sucrose-epichlorohydrin copolymers.

[0100] Commercially available sucrose-epichlorohydrin copolymers can be used. For example, Ficol® from Cytiva can be suitably used, and more specifically, Ficol® PM 70 and Ficol® PM 400 can be used.

[0101] Examples of cellulose derivatives include methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and hydroxyethylcellulose, with methylcellulose being preferred.

[0102] The weight-average molecular weight of the water-soluble polymer is preferably 600 or more, more preferably 1,000 or more, and even more preferably 2,000 or more. On the other hand, low molecular weight compounds with a weight-average molecular weight of about 200 may reduce the efficiency of polynucleotide encapsulation in the capsid. The upper limit of the weight-average molecular weight of the water-soluble polymer is not particularly limited and may be, for example, 500,000 or less, 1,000,000 or less, or 4,000,000 or less. That is, the weight-average molecular weight of the water-soluble polymer may be 600 to 4,000,000, 1,000 to 1,000,000, or 2,000 to 500,000.

[0103] If the composition contains polyethylene glycol, its number average molecular weight is preferably 200 or more, more preferably 600 or more, more preferably 1,000 or more, even more preferably 2,000 or more, and particularly preferably 4,000 or more. Furthermore, the number average molecular weight of polyethylene glycol is preferably 4,000,000 or less, more preferably 1,000,000 or less, even more preferably 100,000 or less, even more preferably 20,000 or less, and particularly preferably 10,000 or less. In other words, the number average molecular weight of polyethylene glycol may be 200 to 4,000,000, 600 to 1,000,000, 1,000 to 100,000, 2,000 to 20,000, or 4,000 to 10,000. Furthermore, the values ​​may be 600 to 4,000,000, 1,000 to 4,000,000, 1,000 to 20,000, 2,000 to 20,000, or 4,000 to 20,000.

[0104] If the composition contains a water-soluble polymer, its concentration is not particularly limited, but may be 0.01% (w / v) or more, 0.03% (w / v) or more, or 0.1% (w / v) or more. It may also be 20% (w / v) or less, 15% (w / v) or less, 10% (w / v) or less, or 5% (w / v) or less. In other words, the concentration of the water-soluble polymer may be 0.01 to 20% (w / v), 0.03 to 15% (w / v), 0.1 to 10% (w / v), or 0.1 to 5% (w / v). The concentration of the water-soluble polymer can be appropriately set according to the molecular weight of the water-soluble polymer. In particular, when the weight-average molecular weight or number-average molecular weight of the water-soluble polymer is 1,000,000 or more, the concentration may be 0.001 to 1% (w / v), 0.01 to 0.5% (w / v), or 0.01 to 0.1% (w / v). When the concentration of the water-soluble polymer is within the above range, the molecular crowding effect is more easily exerted, and the efficiency of encapsulation of polynucleotides into the capsid may improve. Furthermore, when the concentration of the water-soluble polymer is within the above range, even at high concentrations of monovalent cation salts of 100 to 400 mM, and especially 200 to 400 mM, the formation of Rep protein polymers and complexes with capsids and polynucleotides is less likely to be inhibited, making it easier to maintain a high efficiency of encapsulation of polynucleotides into the capsid. In other words, in the encapsulation process, the water-soluble polymer may be mixed so that its concentration is within the above range.

[0105] The composition may contain a surfactant. The surfactant is not particularly limited as long as it has both a hydrophilic group and a lipophilic group, but a nonionic surfactant is preferred. Furthermore, the surfactant is preferably a polyoxyethylene chain. Examples of nonionic surfactants having a polyoxyethylene chain include polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (10) octylphenyl ether, Tween® 20, Triton® X-100, and Pluronic® F-68.

[0106] If the composition contains a surfactant, its concentration is not particularly limited, but may be 0.0001% (w / v) or more, 0.001% (w / v) or more, or 0.01% (w / v) or more. It may also be 3% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, or 0.2% (w / v) or less. That is, the concentration of the surfactant may be 0.0001 to 3% (w / v), 0.001 to 1% (w / v), 0.01 to 0.5% (w / v), or 0.01 to 0.2% (w / v). If the concentration of the surfactant is within the above range, the efficiency of encapsulation of polynucleotides into the capsid may be improved. That is, in the encapsulation step, the surfactant may be mixed so that its concentration is within the above range.

[0107] The composition preferably contains substantially no cell lysate, and more preferably does not contain any cell lysate. The cell lysate may refer to, for example, a solution obtained by disrupting, dissolving, or extracting the first cells or the second cells, or a solution obtained by disrupting, dissolving, or extracting other cells. "Substantially no cell lysate" means that the rAAV particles ultimately produced do not contain impurities derived from host cells such as the first cells or the second cells, and specifically, the amount of cell lysate may be 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.1% by mass or less, or 0.01% by mass or less, based on the total amount of the composition.

[0108] The composition does not need to contain one or more adenovirus helper genes selected from the group consisting of E1A, E1B, E2A, E4, and VA, nor does it need to contain two or more of these, nor does it need to contain any of them. In other words, the composition does not need to contain E1A, E1B, E2A, E4, and VA. Furthermore, it is preferable that the composition contains substantially only capsid and Rep protein as proteins. "Containing substantially only capsid and Rep protein as proteins" means that the proportion of capsid and Rep protein among the proteins contained in the composition is 90% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, 99.9% by mass or more, or 99.99% by mass or more. The fact that the composition contains substantially only capsid and Rep protein as proteins can be rephrased as the proteins contained in the composition being substantially composed of capsid and Rep protein.

[0109] The temperature in this process, i.e., the temperature of the composition, should be sufficient to activate the Rep protein. Such a temperature is, for example, 20 to 55°C, preferably 27 to 45°C, and more preferably 32 to 45°C.

[0110] When the capsid and the Rep protein are mixed with a polynucleotide encoding the target sequence, the polynucleotide is encapsulated in the capsid by the action of the Rep protein. The time required for this process should be sufficient to encapsulate the polynucleotide in the capsid, for example, from 5 minutes to 100 hours.

[0111] The manufacturing method of this embodiment may include a reaction termination step after the step of encapsulating the polynucleotide in the capsid. Examples of reaction termination steps include high-temperature treatment (e.g., 95°C), deactivation of the Rep protein by adding an acid, alkali, or organic solvent, and removal of divalent metal ions by adding a chelating agent such as ethylenediaminetetraacetic acid (EDTA). Another method involves immobilizing the Rep protein on a carrier, carrying out the reaction, and removing the Rep protein from the reaction solution by membrane separation.

[0112] The manufacturing method of this embodiment may include a step of purifying the rAAV particles after the step of encapsulating the polynucleotide in the capsid. Examples of purification methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, size exclusion chromatography, isoelectric focusing precipitation, centrifugation, ultracentrifugation, dialysis, filter separation, reversed-phase chromatography, and ion pair chromatography. These methods may be used individually or in appropriate combinations of two or more methods. The purification of the rAAV particles can be performed to a desired degree.

[0113] The rAAV particles produced by the manufacturing method of this embodiment can be used to introduce polynucleotides encapsulated in a capsid into cells by infecting them. Furthermore, the polynucleotides introduced by the rAAV particles can be used as knock-inners for genome editing technologies such as the ZFN system, TALEN system, and CRISPR / Cas system. That is, another embodiment of the present invention is a method for incorporating a target sequence into the genome of a cell, comprising the steps of infecting a cell with rAAV particles and cleaving the genome of the cell, wherein the rAAV particles contain polynucleotides encoding the target sequence. The method will be described in detail below.

[0114] The step of cutting the genome of the cell may be performed by genome editing technology. Examples of genome editing technologies include one or more selected from the group consisting of the ZFN system, the TALEN system, and the CRISPR / Cas system. Methods for cutting the genome of the cell by genome editing technology include using a sequence-specific endonuclease or introducing the sequence-specific endonuclease into the cell. Examples of sequence-specific endonucleases include ZFN, TALEN, Cas9, etc. If the genome editing technology is the CRISPR / Cas system, a guide RNA (gRNA) containing a sequence complementary to the target sequence may be further introduced into the cell.

[0115] The above description applies to the polynucleotide encoding the target sequence. The target sequence is not particularly limited, but preferably includes a sequence that is completely or substantially complementary to the ends produced by the process of cutting the cell genome.

[0116] In the method according to this embodiment, it is preferable that the polynucleotide encoding the target sequence does not contain some or all of the ITR at its terminus. Knock-inners for genome editing are considered to be more efficient with single-stranded DNA than with double-stranded DNA (see, for example, Bennett H et al. (Methods. 2021 Jul;191:3-14)). When the DNA encapsulated in the rAAV particle has an ITR at its terminus, the self-associated ITR acts like a primer, synthesizing a complementary strand of the DNA and forming double-stranded DNA. Therefore, in this case, the knock-in efficiency is considered to be inferior to that of a polynucleotide that does not contain an ITR.

[0117] As described above, the present inventors have found that rAAV particles can be produced by mixing a capsid and a Rep protein with a polynucleotide encoding a target sequence, and have completed the present invention. That is, another embodiment of the present invention is a method for producing recombinant adeno-associated virus particles, comprising the step of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid, wherein the composition produced by the mixing in the step comprises substantially only a capsid and a Rep protein as proteins. However, this does not prevent the composition from containing BSA as a stabilizer. That is, the composition may consist substantially only of a capsid, a Rep protein, and BSA as proteins. Another embodiment of the present invention is a method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, thereby encapsulating the polynucleotide in the capsid, wherein the composition resulting from the mixing in the step is free of E1A, E1B, E2A, E4, and VA. In this embodiment, the capsid, Rep protein, polynucleotide encoding a target sequence, and composition are described herein by reference.

[0118] The present invention will be described more specifically below based on examples, but the present invention is not limited to these examples.

[0119] [Experimental Example 1] DNA Embedding in Capsid 1) Preparation of Embedding Substrate An AAV2 vector (VectorBuilder) containing single-stranded DNA having the sequence shown in SEQ ID NO: 56 or its complementary strand sequence was incubated at 95°C for 15 minutes to inactivate the capsid protein and release the DNA from within the capsid. The released DNA was separated from the denatured capsid protein using a DNA purification kit (FastGene® Gel / PCR Extraction Kit 300prep) to prepare the vector sequence. The vector sequence contains a gene encoding GFP between two ITR regions. The prepared vector sequence was quantified by qPCR (THUNDERBIRD® Probe qPCR Mix). qPCR was performed using the probe method with forward and reverse primers shown in SEQ ID NOs: 57 and 58, and the probe shown in SEQ ID NO: 59. The standard sample was synthesized by Integrated DNA Technologies using the sequence shown in Sequence ID No. 60, and the concentration was adjusted according to the mole count specified by the company.

[0120] 2) Rep68 Protein Preparation A Rep68 expression vector was prepared by embedding the sequence encoding AAV2 Rep68, which has a histidine tag and GFP protein at its N-terminus, in the NcoI / XhoI site of the pET-15b vector. Hereafter, unless otherwise specified, Rep68 refers to Rep68 derived from AAV2. The sequence of the Rep68 expression vector is shown in Sequence ID No. 61. This vector was used to transform E. coli BL21 (DE3) strain to construct a Rep68 expression strain. The Rep68 expression strain was cultured in 8 L of LB medium at 37°C until the OD was 0.4, and then isopropyl-β-D(-)-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and Rep68 expression was induced at 37°C for 3 hours. After the culture was completed, the bacterial cells were collected as a pellet from the culture medium by centrifugation.

[0121] Rep68 was purified using buffers A, B, C, and D having the following compositions. The details are described below. The bacterial pellet was suspended in 267 mL of buffer A, and the suspension was sonicated for 20 minutes using an ISONATOR 201M (KUBOTA). The lysate was centrifuged at 12,000 × g for 20 minutes at 4°C, the supernatant was collected, and bacterial residue was removed. The obtained supernatant was affinity purified using AKTA Pure (GE Healthcare Life Sciences) and HisTALON Superflow Cartridge 5 mL (Takara Bio), with buffer A as the equilibration buffer and buffer B as the elution buffer. To 5 mL of the eluted fraction containing Rep68, PreScience® Protein (Cytiva) was added at a final concentration of 1 unit / mL, and incubated at 4°C for 16 hours to remove the histidine tag and GFP protein from Rep68. The solution treated with PreScience® Protein was buffer-exchanged to Buffer A using HiPrep® 26 / 10 Desalting (Cytiva), and then affinity purification was performed again using HisTALON Superflow Cartridge 5 mL (Takara Bio), and the clear fraction was recovered. The pass-through fraction was subjected to size exclusion chromatography using C buffer and HiLoad 16 / 600 Superdex 200 prep grade (Cytiva). A 15 mL gel elution fraction was obtained from the fraction in which a band for Rep68 protein was confirmed by SDS-PAGE. This fraction was concentrated to 250 μL using Amicon Ultra-4 10 kDa (Merck Millipore), and 250 μL of D buffer was added to obtain purified Rep68 protein. The purified Rep68 protein was stored at -80°C. The concentration of Rep68 protein was quantified from the band intensity by SDS-PAGE. BSA was used as the calibration curve.

[0122] Buffer A: 20 mM Tris-HCl, 500 mM NaCl, 10 mM Imidazole, 10% (w / v) Glycerol, 1 mM Dithiothreitol, pH 7.9 (4°C) Buffer B: 20 ​​mM Tris-HCl, 500 mM NaCl, 300 mM Imidazole, 10% (w / v) Glycerol, 1 mM Dithiothreitol, pH 7.9 (4°C) Buffer C: 20 mM Tris-HCl, 200 mM NaCl, 1 mM Dithiothreitol, pH 7.9 (4°C) Buffer D: 20 mM Tris-HCl, 200 mM NaCl, 50% (w / v) Glycerol, 1 mM Dithiothreitol, pH 7.9 (4°C)

[0123] 3) DNA encapsulation reaction into capsids: DNA encapsulation into capsids was performed using a vector sequence, purified Rep68 protein, and AAV2 empty capsids (VectorBuilder). The reaction solution consisted of 25 mM HEPES-KOH and 5 mM MgCl. ２ The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 0.2 nM AAV2 empty capsid, 1 nM inclusion sequence, 200 nM Rep68, and pH 7.5. The reaction volume was 10 μL, and the mixture was reacted at 37°C for 1 hour using a thermal cycler. A sample without Rep68 was used as a comparative example.

[0124] 4) DNase (c-LEcta) was added to the reaction solution for detection of embedded sequences by qPCR to a final concentration of 80,600 U / mL, and incubated at 37°C for 1 hour to remove DNA outside the capsid. 5 μL of this DNase-treated solution was then mixed with 5 μL of 10 mM EDTA to inactivate the DNase, followed by heating at 95°C for 15 minutes to denature the capsid and DNase. The inactivation solution was then diluted with 20 μL of pure water, and 5 μL of this solution was subjected to qPCR using THUNDERBIRD® Probe qPCR Mix. qPCR was performed using the probe method, employing the forward and reverse primers shown in SEQ ID NOs. 57 and 58, and the probe shown in SEQ ID NO. 59.

[0125] 5) Results The concentration (vg / μL) of the vector sequence encapsulated in the capsid is shown in Figure 1. Under the conditions in which Rep68 was added, the concentration of the vector sequence was 1.18 × 10⁶. ６ Under conditions where vg / μL was detected and Rep68 was not added, the vector sequence was 1.67 × 10⁶. ３ It was detected at vg / μL. In other words, the addition of Rep68 promoted DNA inclusion in the capsid by approximately 1,000 times.

[0126] [Experimental Example 2] Effect of the presence or absence of the ITR region on DNA encapsulation efficiency 1) Preparation of encapsulation substrate The template DNA shown in SEQ ID NO: 62 was amplified by PCR using the forward primer shown in SEQ ID NO: 63 and the reverse primer shown in SEQ ID NO: 64 to obtain a DNA fragment. This DNA fragment was used as the blunt-end sequence (SEQ ID NO: 65). This blunt-end sequence was treated with restriction enzymes using BamHI (Takara Bio Inc.) to form an overhanging end, which was used as the overhanging end sequence (SEQ ID NO: 66). The 4 bases at the 5' end of the overhanging end sequence formed the overhanging end. Eurofins Genomics was commissioned to synthesize single-stranded DNA containing the ITR region derived from AAV2, with the sequences shown in SEQ ID NOs: 67 and 68. The 5' end of the DNA with the sequence shown in SEQ ID NO: 68 was phosphorylated with T4 polynucleotide kinase and then annealed with the DNA with the sequence shown in SEQ ID NO: 67. The ITR-containing sequence (SEQ ID NO: 69) was obtained by ligating the aforementioned overhanging end sequence to this double-stranded DNA using T3 DNA ligase (New England Biolabs). The three prepared sequences were quantified by qPCR (THUNDERBIRD® Probe qPCR Mix). qPCR was performed using the probe method with the forward and reverse primers shown in SEQ ID NOs: 70 and 71, and a probe modified with FAM® dye at the 5' end and TAMRA® quencher at the 3' end of the sequence shown in SEQ ID NO: 72. The standard was synthesized by Integrated DNA Technologies using the sequence shown in SEQ ID NO: 73, and the concentration was adjusted according to the mole count specified by the company.

[0127] 2) DNA inclusion reaction in capsid: Purified Rep68 protein was obtained in the same manner as in Experimental Example 1. The DNA inclusion reaction in capsid was performed in the same manner as in Experimental Example 1, except that one of the three inclusion sequences shown in SEQ ID NOs. 65, 66, and 69 (blunt-end sequence, overhang-end sequence, or ITR-containing sequence) was used instead of the vector sequence.

[0128] 3) Detection of inclusion sequences by qPCR QPCR was performed in the same manner as in Experimental Example 1, except that the forward and reverse primers shown in SEQ ID NOs. 70 and 71 were used, and a probe modified with FAM® dye at the 5' end and TAMRA® quencher at the 3' end of the sequence shown in SEQ ID NO. 72 was used.

[0129] 4) The concentrations (vg / μL) of each sequence encapsulated in the resulting capsid are shown in Figure 2. Under the condition of adding Rep68, the concentration was 1.0 × 10⁻¹⁶ for all blunt-end sequences, overhang-end sequences, and ITR-containing sequences. ５ Accumulation of vg / μL or higher was confirmed, and the accumulation was approximately 10,000 times greater compared to the condition without Rep68 addition.

[0130] [Experimental Example 3] Comparison of serotypes of capsids used to encapsulate DNA sequences The capsids that make up AAV exhibit diversity in protein sequences among serotypes. We investigated whether DNA sequence encapsulation using Rep protein was possible with serotypes other than AAV2, which was used in Experimental Examples 1 and 2. Empty capsids derived from AAV1, AAV2, or AAV6 (all from Cosmo Bio Co., Ltd.) were used as empty capsids, and ITR-containing sequences were used as encapsulation sequences. DNA encapsulation experiments were conducted under the same conditions as in Experimental Example 1. The results are shown in Figure 3.

[0131] As a result, similar to AAV2, AAV1 and AAV6 also showed a result of 5.0 × 10⁶ when Rep68 was added. ５ Accumulation of rAAV particles at levels above vg / μL was confirmed, and the accumulation was more than 1,000 times greater compared to the condition without Rep68 addition.

[0132] [Experimental Example 4] Inclusion of DNA Fragments Containing Modified Residues Naturally occurring AAV genomes and the DNA sequences loaded into conventionally used transfer vectors are unmodified polynucleotides. However, it is expected that introducing modified residues into the inclusion sequence can impart functions such as stability to rAAV particles. Therefore, we investigated the inclusion of DNA fragments containing 5-methylcytosine, 2'-O-methyl, 2'-O-methoxyethyl, 2'-F, phosphorothioate DNA (hereinafter also referred to as "PS-DNA"), or RNA as modified residues. The synthesis of the inclusion substrate, the inclusion reaction, and the detection of the inclusion sequence by qPCR were carried out in the same manner as in Experimental Example 2. Specifically, the template DNA shown in SEQ ID NO: 62 was PCR amplified using the forward and reverse primers shown in Table 1 to prepare the blunt-end sequences shown in SEQ ID NOs: 88-100. The blunt-end sequence shown in Sequence ID No. 88 is a double-stranded DNA consisting of the sequence shown in Sequence ID No. 88 and its complementary strand, while the other blunt-end sequences are double-stranded DNA or DNA / RNA hybrid strands consisting of the two sequences shown in Table 1. The primers were purchased from Eurofins Genomics. The DNA inclusion reaction into the capsid was performed in the same manner as in Experimental Example 1, except that these blunt-end sequences were used instead of the vector sequence.

[0133]

[0134] Figure 4 shows the concentration (vg / μL) of each DNA fragment encapsulated in the capsid. Under conditions with Rep68 added, the concentration was 6.0 × 10⁻⁶. ５ Accumulation of vg / μL or more was confirmed, and the accumulation was more than 10,000 times greater compared to the condition without Rep68 addition. In other words, according to the method of one embodiment of the present invention, rAAV particles containing DNA fragments with modified residues can be obtained.

[0135] [Experimental Example 5] Encapsidation using GFP-tagged Rep68 protein The Rep68 protein used for the encapsidation reaction in the above Examples was expressed in E. coli as a fusion protein with a GFP region added. This was cleaved with protease to remove GFP and used for subsequent experiments. In this section, it was verified whether the Rep68 fusion protein retaining the GFP region has DNA encapsidation activity. Using an AAV2 empty vector (manufactured by VectorBuilder) as the empty capsid and a blunt-end sequence (SEQ ID NO: 65) as the DNA to be encapsulated, an encapsidation reaction was carried out under the same conditions as in Experimental Example 1. For GFP-tagged Rep68 protein, the fraction obtained after affinity purification in [Experimental Example 1] was quantified by SDS-PAGE (converted to BSA), added at a final concentration of 20 nM, and the reaction was allowed to proceed. As a result, the detected value of encapsidated product was 14.8 vg / μL under the condition where no GFP-tagged Rep68 protein was added, whereas the encapsidated product was 105061.2 vg / μL under the condition where GFP-tagged Rep68 was added, showing a difference of more than 7,000-fold. This indicated that the GFP-added Rep68 protein also has the function of encapsidating a polynucleotide encoding the target sequence into the capsid.

[0136] [Experimental Example 6] Examination of conditions for DNA encapsidation reaction The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 0.1% Pluronic® F-68, 44 mM NaCl, 0.6–9 nM AAV2 empty capsid, 0.3–4.1 nM inclusion sequence (blunt-ended sequence: SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and 30 μL of reaction solution. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The concentration of the inclusion sequence added to the reaction mixture was quantified by digital PCR. Digital PCR was performed using the QIAcuity Digital PCR System (manufactured by Qiagen Co., Ltd.) with the forward and reverse primers shown in SEQ ID NOs: 70 and 71, the probe sequence shown in SEQ ID NO: 72, and the QIAcuity Probe PCR Kit. Subsequently, DNA outside the capsid was removed in the same manner as in Experimental Example 1, and the capsid and DNase were further denatured. These samples were subjected to qPCR in the same manner as in Experimental Example 2 to detect inclusion sequences.

[0137] Figure 5 shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid. Under the conditions of 9 nM AAV2 empty capsid and 4.1 nM inclusion sequence, the inclusion sequence was 2.27 × 10⁶. ９ It was detected at vg / μL, and under the conditions of 0.6 nM AAV2 empty capsid and 0.3 nM inclusion sequence, the inclusion sequence was 3.98 × 10⁶. ７ It was detected at vg / μL. This confirms that the amount of DNA inclusion in the capsid can be increased in a concentration-dependent manner between the empty capsid and the inclusion sequence in the reaction solution.

[0138] [Experimental Example 7] Comparison of serotypes of capsids encapsulating DNA sequences 2. The reaction solution composition is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.05 μg / μL BSA, 0.1% Pluronic® F-68, 60 mM NaCl, 3 nM empty capsid (AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9), 3 nM inclusion sequence (blunt-ended sequence: SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and 30 μL of reaction solution. DNA inclusion in the capsid was performed in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. A sample without the addition of an empty capsid was used as a comparative example. The inclusion sequence was detected by qPCR on these samples in the same manner as in Experimental Example 2.

[0139] Figure 6 shows the concentration (vg / μL) of the inclusion sequence enclosed in the capsid. Under conditions without the addition of an empty capsid, the inclusion sequence was 2.75 × 10⁶. ４ Although vg / μL was detected, the value was similar to that under conditions without Rep68 addition and was considered equivalent to the background signal. On the other hand, under conditions with AAV1 addition, the value was 1.83 × 10⁻⁶. ６ vg / μL, AAV2 is 4.14 × 10 ８ vg / μL, AAV3 is 2.95 × 10 ５ vg / μL, AAV5 is 2.14 × 10 ７ vg / μL, AAV6 is 6.23 × 10 ６ At vg / μL, AAV8 yields 7.63 × 10⁻⁶. ５ vg / μL, AAV9 is 3.12 × 10 ６ An inclusion sequence at vg / μL was detected. This means that DNA inclusion in the capsid was confirmed even in different serotypes.

[0140] [Experimental Example 8] Full capsid rate after DNA encapsulation 1) DNA encapsulation reaction in capsid The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 0.1% Pluronic® F-68, 44 mM NaCl, 9 nM AAV2 empty capsid, 3 nM inclusion sequence (blunt-end sequence: SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and 30 μL of reaction solution. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. A sample without Rep68 was used as a comparative example.

[0141] 2) Measurement of full capsid ratio by mass photometry The sample after the DNA inclusion reaction was diluted 9-fold with Phosphate-Buffered Saline to a capsid concentration of 1 nM and subjected to analysis using SamuxMP (Refeyn).

[0142] 3) Results The results of the mass photometry measurements are shown in Figure 7. Under the condition with Rep68 added, the full capsid rate was 67.9%, while under the condition without Rep68 added, the full capsid rate was 7.8%. In other words, the addition of Rep68 promoted DNA inclusion into the capsid.

[0143] [Experimental Example 9] Evaluation of infectivity of capsids after DNA encapsulation 1) DNA encapsulation reaction in capsids Composition of reaction solution: 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 0.1% Pluronic® F-68, 44 mM NaCl, 10 nM AAV2 empty capsid, 1 nM vector sequence, 100 nM Rep68, pH 7.5, and a volume of 30 μL. DNA inclusion into the capsid was performed in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The vector sequence used was the one prepared in Experimental Example 1. A sample without Rep68 and empty capsid was used as a comparative example. The inclusion sequence was detected by qPCR on these samples in the same manner as in Experimental Example 1.

[0144] 2) Infection experiment on HEK293 cells HEK293 cells were cultured at 37°C and 5% CO2. ２The experiment was conducted under the following conditions. HEK293 cells were seeded in a low-adhesion 96-well plate at a density of 3000 cells / well using FreeStyle® F17 (Gibco), and then swirled in a plate shaker. The Multiplicity of Infection was 1.7 × 10⁶ based on the quantitative value of inclusion capsids by qPCR. ６ The sample after the mounting reaction was added in this manner, and the expression of green fluorescence was analyzed after 72 hours using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0145] 3) Results Under the conditions of 10 nM AAV2 empty capsid and 100 nM Rep68, 99.8% of cells were positive. Under the condition without Rep68, 2.2% of cells were positive, and under the condition without empty capsid, 6.0% of cells were positive. In other words, the infectivity of the full capsid produced by the DNA inclusion reaction was confirmed.

[0146] [Experimental Example 10] The composition of the reaction solution used for the sealing reaction with REP40 and REP68 was 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 1 nM AAV2 empty capsid, 1 nM inclusion sequence (blunt-ended sequence: SEQ ID NO: 65), 0–200 nM Rep68, 0–200 nM Rep40, pH 7.5, and a reaction volume of 30 μL. DNA inclusion into the capsid was performed in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. In each sample, the sum of the concentrations of Rep68 and Rep40 was adjusted to 200 nM. For these samples, qPCR was performed in the same manner as in Experimental Example 2 to detect the inclusion sequence.

[0147] Figure 8 shows the concentration (vg / μL) of the inclusion sequence encapsulated in the capsid. Under conditions of 200 nM Rep40 and 0 nM Rep68, the inclusion sequence was 1.82 × 10⁶. ６ Under the conditions of vg / μL, 160 nM Rep40, and 40 nM Rep68, the result was 6.91 × 10⁻¹⁶. ７ Under the conditions of vg / μL, 120 nM Rep40, and 80 nM Rep68, the result is 1.52 × 10⁻¹⁰.８ Under the conditions of vg / μL, 100nM Rep40, and 100nM Rep68, the result is 2.74 × 10⁻⁶. ８ Under the conditions of vg / μL, 80 nM Rep40, and 120 nM Rep68, the result was 3.18 × 10⁻⁶. ８ Under the conditions of vg / μL, 40 nM Rep40, and 160 nM Rep68, the result is 3.11 × 10⁻¹⁰. ８ Under the conditions of vg / μL, 0nM Rep40, and 200nM Rep68, the result is 2.80 × 10⁻⁶. ８ It was detected at vg / μL. In other words, the encapsulation reaction efficiency was dependent on the Rep68 concentration in the range of 0 to 120 nM.

[0148] [Experimental Example 11] The composition of the plasmid DNA mounting reaction solution was 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 5 mM dithiothreitol, 4 mM ATP, 0.05 μg / μL BSA, 0.1% Pluronic® F-68, 55 mM NaCl, 1 nM AAV2 empty capsid, 1 nM DNA sequence (vector sequence or plasmid DNA), 100 nM Rep68, pH 7.5, and a volume of 30 μL. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The vector sequence used was the one prepared in Experimental Example 1. The base sequence of the plasmid DNA is shown in Sequence ID No. 101. For samples using the vector sequence as the inclusion substrate, qPCR was performed in the same manner as in Experimental Example 1 to detect the inclusion sequence. For samples using plasmid DNA as the inclusion substrate, qPCR was performed in the same manner as in Experimental Example 1, except that the forward and reverse primers shown in SEQ ID NOs. 102 and 103, and the probe shown in SEQ ID NO. 104, modified with FAM® dye at the 5' end and TAMRA® quencher at the 3' end were used.

[0149] Figure 9 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid. Under conditions where the vector sequence was used as the substrate, the concentration was 2.73 × 10⁻¹⁶. ７ It was detected at vg / μL, and under conditions using plasmid DNA as a substrate, the concentration was 3.81 × 10⁶. ５It was detected at vg / μL. In other words, when a vector sequence, which is linear double-stranded DNA, was used as the substrate, the amount of DNA encapsulated in the capsid was approximately 73 times higher compared to when plasmid DNA, which is circular double-stranded DNA, was used.

[0150] [Experimental Example 12] Investigation of combinations of PEG concentration and protein concentration. The composition of the reaction solution was 25 mM HEPES-KOH and 5 mM MgCl. ２ 5 mM dithiothreitol, 4 mM ATP, 0.05 μg / μL BSA, 0.1% Pluronic® F-68, 50 mM NaCl, 1 nM AAV2 empty capsid, 1 nM DNA sequence (blunt-ended sequence: SEQ ID NO: 65), 0.5–5% (w / v) polyethylene glycol 6,000 (PEG6,000, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 169-09125), 20–200 nM Rep68, pH 7.5, reaction volume 20 μL. DNA inclusion reaction into capsids was carried out in the same manner as in Experimental Example 1, except that the above reaction solution was reacted for 1 hour at 37°C using a thermal cycler. qPCR was performed on these samples in the same manner as in Experimental Example 2 to detect the inclusion sequences.

[0151] Table 2 shows the concentration of DNA sequences encapsulated in the capsid (vg / μL) for each combination of PEG concentration and Rep concentration. It was found that, in all Rep concentration conditions, the concentration of DNA encapsulated in the capsid increased with the addition of PEG 6,000 compared to the condition without PEG 6,000, indicating that the addition of PEG improved reactivity.

[0152] [Experimental Example 13] Reaction at high salt concentration under PEG addition conditions 1 The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 65–245 mM NaCl, 0% or 3% (w / v) PEG6,000, 10 nM AAV2 empty capsid, 10 nM DNA sequence (blunt-ended sequence: SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and 30 μL of reaction solution. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The inclusion sequences were detected by qPCR on these samples in the same manner as in Experimental Example 2. In addition to the NaCl added to the reaction solution, ATP, Rep68, and the empty capsid also contain NaCl or KCl, and the total monovalent cation concentration of these is estimated to be between 128 and 308 mM.

[0153] The concentration of the DNA sequence encapsulated in the capsid (vg / μL) is shown for each total monovalent cation concentration. Figure 10 shows the condition without PEG addition, and Figure 11 shows the condition with 3% PEG addition. Under the condition without PEG6,000 addition, the concentration of the DNA sequence encapsulated in the capsid decreased as the total monovalent cation concentration increased from 128 mM. On the other hand, under the condition with 3% PEG6,000 addition, the concentration of the DNA sequence encapsulated in the capsid was highest at a total monovalent cation concentration of 195 mM, suggesting that the addition of PEG6,000 provided cation concentration tolerance. Furthermore, even when comparing under the condition with a total monovalent cation concentration of 128 mM, the concentration of the DNA sequence encapsulated in the capsid increased by approximately eight times with the addition of PEG6,000, improving encapsulation efficiency.

[0154] [Experimental Example 14] Reaction at high salt concentration under PEG addition conditions 2 The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 125–215 mM NaCl, 3%–10% (w / v) PEG6,000, 10 nM AAV2 empty capsid, 10 nM DNA sequence (blunt-ended sequence: SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and a reaction volume of 30 μL. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. For these samples, qPCR was performed in the same manner as in Experimental Example 2 to detect the inclusion sequence. In addition to the NaCl added to the reaction solution, ATP, Rep68, and the empty capsid also contain NaCl or KCl, and the total monovalent cation concentration of these is estimated to be between 188 and 278 mM. The total monovalent cation concentration shown in Figure 12 is the sum of these values.

[0155] Figure 12 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each PEG concentration and total monovalent cation concentration. Under the PEG 3% condition, the concentration of the DNA sequence encapsulated in the capsid decreased as the total monovalent cation concentration increased from 188 mM. On the other hand, at higher PEG concentrations of 5%, 7%, and 10%, the concentration of the DNA sequence remained high even at high total monovalent cation concentrations. Within these experimental conditions, the highest concentration of the DNA sequence encapsulated in the capsid was observed under the conditions of 5% PEG 6,000 and a total monovalent cation concentration of 248 mM, at 5.6 × 10⁻¹⁶. ９ The concentration was vg / μL, indicating that DNA was encapsulated in 94% of the added empty capsids.

[0156] [Experimental Example 15] Reaction at high salt concentration under PEG addition conditions 3 The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 180–330 mM NaCl, 5%–10% (w / v) PEG6,000, 10 nM AAV2 empty capsid, 10 nM DNA sequence (blunt-ended sequence: SEQ ID NO: 65), 0 nM or 100 nM Rep68, pH 7.5, and 30 μL of reaction solution. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The inclusion sequences were detected by qPCR on these samples in the same manner as in Experimental Example 2. In addition to the NaCl added to the reaction solution, ATP, Rep68, and the empty capsid also contain NaCl or KCl, and the total monovalent cation concentration of these is estimated to be between 243 and 393 mM. The total monovalent cation concentration shown in Figure 13 is the sum of these values.

[0157] Figure 13 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each PEG concentration and total monovalent cation concentration. Under the condition of a PEG 6,000 concentration of 5%, the activity was highest at a total monovalent cation concentration of 293 mM. On the other hand, under the condition of a higher PEG concentration of 10%, the concentration of the DNA sequence was high even at high total monovalent cation concentrations. Under the condition of 10% PEG 6,000, the concentration of the DNA sequence encapsulated in the capsid was highest at a total monovalent cation concentration of 343 mM, and activity was maintained even at 393 mM. Furthermore, under the condition without the addition of Rep, the concentration of the DNA sequence was below the detection limit, confirming that the encapsulation reaction was functioning.

[0158] [Experimental Example 16] Study of polyethylene glycol molecular weight. The reaction solution composition was 25 mM HEPES-KOH and 5 mM MgCl. ２The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 75 mM, 125 mM or 175 mM NaCl, polyethylene glycol of various molecular weights, 1 nM AAV2 empty capsid, 1 nM DNA sequence (SEQ ID NO: 65), 100 nM Rep68, pH 7.5, and 24 μL of reaction solution. The final concentrations of polyethylene glycol of each molecular weight in this reaction solution were 1% (w / v), 3% (w / v), or 10% (w / v) for polyethylene glycol 200 (PEG200), polyethylene glycol 600 (PEG600), polyethylene glycol 1,000 (PEG1,000), polyethylene glycol 2,000 (PEG2,000), polyethylene glycol 4,000 (PEG4,000), polyethylene glycol 6,000 (PEG6,000), and polyethylene glycol 20,000 (PEG20,000), and 0.03% (w / v), 0.01% (w / v), or 0.3% (w / v) for polyethylene glycol 4,000,000 (PEG4,000,000). All polyethylene glycols were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. DNA inclusion in capsids was performed in the same manner as in Experimental Example 1, except that the reaction solution was reacted at 37°C for 1 hour using a thermal cycler. These samples were subjected to qPCR in the same manner as in Experimental Example 2 to detect the inclusion sequences.

[0159] Table 3 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each PEG concentration and for each monovalent cation concentration. The monovalent cation concentration was calculated based on the concentrations of potassium and sodium ions contained in HEPES-KOH, NaCl, AAV2 empty capsid, and Rep68. Under conditions without polyethylene glycol addition, the concentration of the DNA sequence encapsulated in the capsid was a maximum of 2.1 × 10⁻⁶. ８While the concentration was vg / μL, under conditions where all polyethylene glycols except PEG200 were added, the concentration of the DNA sequence encapsulated in the capsid was higher. In other words, it was confirmed that adding polyethylene glycols with a number-average molecular weight of 600 to 4,000,000 increased the reaction activity of the encapsulation reaction.

[0160] [Experimental Example 17] Reaction under conditions of water-soluble polymer addition: The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 75 mM NaCl, various water-soluble polymers, 1 nM AAV2 empty capsid, 1 nM DNA sequence (SEQ ID NO: 65), 0 nM or 100 nM Rep68, and pH 7.5, with a reaction volume of 30 μL. The final concentrations of water-soluble polymers in this reaction solution were 1% (w / v), 3% (w / v), and 10% (w / v) for Ficol™ PM70, Ficol™ PM400, and Dextran 40,000 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.2% (w / v), 0.6% (w / v), and 2% (w / v) for Polyvinylpyrrolidone K 30 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.03% (w / v), 0.1% (w / v), or 0.3% (w / v) for Polyvinylpyrrolidone K 90 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and Methylcellulose 4,000 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The DNA encapsulation reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction solution was reacted for 1 hour at 37°C using a thermal cycler. For these samples, qPCR was performed in the same manner as in Experimental Example 2 to detect inclusion sequences.

[0161] Table 4 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid. For each water-soluble polymer, there were conditions in which the concentration of the DNA sequence encapsulated in the capsid was higher than under conditions without the addition of the polymer. In other words, it was confirmed that the addition of the water-soluble polymer increased the reaction activity of the encapsulation reaction.

[0162] [Experimental Example 18] Reaction under surfactant addition conditions: The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 75 mM NaCl, various surfactants, 1 nM AAV2 empty capsid, 1 nM DNA sequence (SEQ ID NO: 65), 0 nM or 100 nM Rep68, and pH 7.5, with a reaction volume of 30 μL. The final concentrations of the surfactants in this reaction mixture were 0.001% (w / v), 0.01% (w / v), 0.1% (w / v), or 0.5% (w / v) for polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (10) octylphenyl ether, Tween® 20, Triton® X-100, and Pluronic® F-68. DNA inclusion in capsids was performed in the same manner as in Experimental Example 1, except that the reaction solution was reacted at 37°C for 1 hour using a thermal cycler. These samples were subjected to qPCR in the same manner as in Experimental Example 2 to detect the inclusion sequences.

[0163] Figure 14 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each surfactant concentration under the condition where 100 nM Rep68 was added. Under the condition where Rep68 was not added, the concentration of the DNA sequence was 10 ５ The concentration was less than vg / μL. In all cases, the concentration of the DNA sequence encapsulated in the capsid was higher than in the no-surfactant condition. In other words, it was confirmed that the addition of surfactants increased the reaction activity of the encapsulation reaction.

[0164] [Experimental Example 19] Reaction under conditions of adding cell lysate 1) Preparation of cell lysate without helper factors HEK293 cells were suspended in buffer and homogenized to prepare cell lysate. Specifically, the following procedure was performed: HEK293 cells were heated at 37°C and 5% CO2 ２ Under these conditions, the cells were cultured in a circular motion using a plate shaker with an Erlenmeyer flask and FreeStyle® F17 (Gibco). 2.5–3.5 × 10 ６HEK293 cells, grown to cells / mL, were collected in 10 mL portions and centrifuged at 4000 x g for 15 minutes to remove the cells. The cells were resuspended in 10 mL of PBS and centrifuged at 4000 x g for 15 minutes, then centrifuged in 5 mL of hypotonic buffer (20 mM HEPES pH 7.4, 5 mM KCl, 1.5 mM MgCl). ２ The cells were suspended in 1 mM dithiothreitol and centrifuged at 4000 × g for 15 minutes to obtain the cells before disruption. 2 mL of hypotonic buffer was added to these cells, and the mixture was left to stand on ice for 10 minutes, followed by Downs homogenization. The lysates were centrifuged at 15,000 × g for 15 minutes, and the supernatant was collected as the hypotonic buffer fraction. This hypotonic buffer was diluted 10-fold with storage buffer (20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 25 mM NaCl, 10% Glycerol, 1 mM dithiothreitol) to obtain the storage buffer fraction.

[0165] 2) Preparation of cell lysates containing helper factors HEK293 cells were co-transfected with the pEMBR-1.2 adenovirus helper plasmid described in JP 2024-518553 A using the Transfection reagent PEI MAX. Four days after transfection, 10 mL of cultured cells were collected, and cell lysates were prepared in the same manner as in 1) above, consisting of a hypotonic buffer fraction and a storage buffer fraction.

[0166] 3) DNA encapsulation reaction in capsids: Final concentrations during the reaction are 25 mM HEPES-KOH and 5 mM MgCl. ２ To 15 μL of a reaction solution fraction prepared to contain 1 mM dithiothreitol, 4 mM ATP, 0.2 μg / μL BSA, 1.0 nM AAV2 empty capsid, 1 nM inclusion sequence (SEQ ID NO: 65), 100 nM Rep68, and pH 7.5, 15 μL of the hypotonic buffer fraction or storage buffer fraction prepared in 1) and 2) was added, and the mixture was reacted at 37°C for 1 hour using a thermal cycler. The fraction without disrupted cells was used as the control.

[0167] Figure 15 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each experimental group. In the hypotonic buffer fraction, the concentration of the DNA sequence was lower than in the fraction without disrupted cells, regardless of the presence or absence of helper factors. Similarly, in the storage buffer fraction, the concentration of the DNA sequence was also lower than in the fraction without disrupted cells, regardless of the presence or absence of helper factors. Therefore, it was considered that components contained in the disrupted cells inhibited the DNA encapsulation reaction in the capsid. In other words, it was confirmed that the reaction solution substantially free of cell disruption solution is useful in this reaction.

[0168] [Experimental Example 20] Examination of Inclusion Reaction Using Rep Protein Homologs 1) Preparation of Rep78 Protein Derived from Each Homolog The sequence encoding Rep78 of an AAV having a histidine tag and MBP protein at the N-terminus was inlaid in the NcoI / XhoI site of the pET-15b vector to create expression vectors for Rep78 derived from each homolog. The serotypes of the AAVs from which each homolog is derived are AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV12, or AAV13 (Sequence IDs 36, 41-44, 46, 47, 50, and 51 respectively). The sequences of these Rep78 expression vectors are shown as Sequence IDs (Sequence IDs 105-113). This vector is E. Coli BL21 (DE3) strain was transformed to construct Rep78-expressing strains derived from each homolog. These Rep78-expressing strains were cultured in 250 mL of LB medium at 37°C until the OD reached 0.3. Isopropyl-β-D(-)-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM, and Rep78 expression from each homolog was induced at 16°C for 16 hours. After the culture period, the bacterial cells were collected as a pellet from the culture medium by centrifugation.

[0169] Rep78 from each homolog was purified using the D buffer described in Experimental Example 1, as well as the E buffer and F buffer having the following compositions. The details are described below. The bacterial pellet was suspended in 10 mL of E buffer, and the suspension was sonicated for 20 minutes using an ISONATOR 201M (KUBOTA). The lysate was centrifuged at 15,000 × g for 10 minutes at 4°C, the supernatant was collected, and bacterial residue was removed. The obtained supernatant was affinity purified using AKTA Pure (GE Healthcare Life Sciences) and HisTALON Superflow Cartridge 5 mL (Takara Bio), with E buffer as the equilibration buffer and F buffer as the elution buffer. To 0.5 mL of the elution fraction containing Rep78, 8.27–39.53 μL of PreScission® Protease (Cytiva, Inc.) (2000 units / mL) was added according to the amount of protein obtained, and the mixture was incubated at 4°C for 16 hours to remove the histidine tag and MBP protein from Rep78. An equal volume of D buffer was added to the solution treated with PreScission® Protease (Cytiva, Inc.) (2000 units / mL) to obtain purified Rep78 protein from each homolog. These purified Rep78 proteins were stored at -80°C. The concentration of these purified Rep78 proteins was quantified from band intensity by SDS-PAGE. BSA was used as the calibration curve.

[0170] E Buffer: 20 mM Tris-HCl, 500 mM NaCl, 20% (w / v) glycerol, 1 mM dithiothreitol, pH 7.9 (4°C) F Buffer: 20 mM Tris-HCl, 350 mM NaCl, 10 mM maltose, 20% (w / v) glycerol, 1 mM dithiothreitol, pH 7.9 (4°C)

[0171] 3) DNA encapsulation reaction in capsid: The reaction solution is composed of 25 mM HEPES-KOH and 5 mM MgCl. ２The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 75 mM NaCl, 10 nM each AAV empty capsid homolog, 1.5 nM inclusion sequence (SEQ ID NO: 65), 250 nM each Rep78 homolog, pH 7.5, and a reaction volume of 30 μL. The DNA inclusion reaction into the empty capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. The AAV serotypes from which each AAV empty capsid homolog was derived were AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, or AAV9. These capsids are encoded by the cap genes shown in SEQ ID NOs: 29-35, respectively. Samples without the addition of empty capsids and Rep protein were used as comparative examples. For these samples, qPCR was performed in the same manner as in Experimental Example 2 to detect inclusion sequences.

[0172] Tables 5 and 6 show the concentration (vg / μL) of the DNA sequence encapsulated in the capsid. In all combinations of Rep78 homolog and AAV empty capsid homolog, the concentration of the DNA sequence was higher compared to samples without the addition of empty capsid and Rep protein. This result suggests that the interaction between the capsid and Rep protein is not limited to specific serotype combinations, but is based on conserved structural or functional characteristics among AAVs.

[0173] [Experimental Example 21] Examination of DNA Chain Length for Inclusion 1) Preparation of Inclusion DNA Plasmid DNA of Sequence ID No. 114 (created by Genscript Inc.) was used as a template for PCR to amplify the sequence to the desired length. After agarose gel electrophoresis, the linear double-stranded DNA to be inclusion was prepared by excision and purification from the agarose gel. Note that Plasmid DNA of Sequence ID No. 114 does not have ITR. The sequence number of the DNA to be inclusion, the base length, and the sequence numbers of the primers are shown in Table 7. The PCR reaction mixture was composed of 30 μL PrimeSTAR® Max DNA Polymerase (Takara Bio), reacted with 50 pg plasmid DNA and 0.5 μM primer in 60 μL. The reaction mixture was subjected to electrophoresis using 1% (w / v) or 2% (w / v) agarose gel, and excision and purification were performed from the agarose gel. The excised gel was gel-extracted using FastGene® Gel / PCR Extraction Kit 300prep, ultimately yielding 30 μL of amplified DNA. qPCR was performed on the prepared inclusion DNA using the probe method, as in Experimental Example 1. The standard sample was the sequence shown in Sequence ID No. 60.

[0174] 2) The composition of the reaction solution for the sealing reaction is 25 mM HEPES-KOH and 5 mM MgCl ２ The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, Pluronic® F-68, 3% (w / v), PEG 6,000, 175 mM NaCl, 4 nM AAV2 empty capsid, 2 nM of each inclusion sequence, 100 nM Rep68, pH 7.5, and 30 μL. DNA inclusion in the empty capsid was performed in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. A sample without Rep protein added under conditions using 2 kbp DNA (sequence 4) was used as a negative control. These samples were subjected to qPCR using the probe method in the same manner as in Experimental Example 1. The standard was the sequence shown in Sequence ID No. 60. As a result, all inclusion sequences showed a negative value of 4.0 × 10⁶ compared to the negative control. ３ The concentration of DNA sequences was higher compared to vg / μL (Figure 16).

[0175] [Experimental Example 22] Comparison of Inclusion End Sequences 1) Preparation of Inclusion DNA The desired length of sequence was amplified by PCR using plasmid DNA of sequence number 114 as a template. After agarose gel electrophoresis, the sequence was excised from the agarose gel and purified to prepare the linear double-stranded DNA to be inclusion. The sequence numbers, base lengths, and primer sequence numbers of the inclusion DNA are shown in Table 8. qPCR was performed on the prepared DNA using the probe method, as in Experimental Example 1. The standard sample was the sequence shown in sequence number 60.

[0176] 2) The composition of the reaction solution for the sealing reaction is 25 mM HEPES-KOH and 5 mM MgCl ２ The reaction mixture consisted of 0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, Pluronic® F-68, 3% (w / v) PEG6,000, 175 mM NaCl, 4 nM AAV2 empty capsid, 2 nM of each inclusion sequence, 100 nM Rep68, pH 7.5, and 30 μL. The DNA inclusion reaction into the empty capsid was carried out in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. A sample without Rep protein added under conditions using 2 kbp DNA was used as a negative control. These samples were subjected to qPCR using the probe method in the same manner as in Experimental Example 1. The standard was the sequence shown in Sequence ID No. 60.

[0177] As a result, all inclusion sequences showed a negative control of 4.0 × 10⁶. ３ The concentration of DNA sequences was higher compared to vg / μL (Figure 17). Furthermore, the ratio of sequence 12, which had the highest DNA sequence concentration, to sequence 8, which had the lowest concentration, was 1.47 times, indicating that the inclusion activity is less affected by the terminal sequence of the inclusion DNA, and that any sequence can be inclusioned.

[0178] [Experimental Example 23] Comparison of the purity of inclusion reactions and their products using plasmid DNA or linear double-stranded DNA containing ITR sequences When using AAV as a gene vector for DNA delivery, it is extremely important that only the intended sequence is inclusion. As confirmed in Experimental Example 12, it is possible to inclusion the target sequence in the capsid even when using plasmid DNA containing the target sequence, but there is a possibility that sequences other than the target sequence from the plasmid DNA sequence will also be inclusion in the capsid. Therefore, in this experimental example, the effect of the DNA structure used in the inclusion reaction on the purity of the DNA inclusion in the AAV vector obtained after the inclusion reaction was evaluated. We investigated whether there was a difference in the proportion of the target sequence inclusion when linear double-stranded DNA consisting only of the sequence shown in SEQ ID NO: 154, the target sequence, was used as the inclusion sequence, and when plasmid DNA (SEQ ID NO: 155) containing the sequence shown in SEQ ID NO: 154, the target sequence, was used as the inclusion sequence. Note that the target sequence has ITRs at both ends. Hereinafter, in this experimental example, the region of the plasmid DNA sequence other than the above target sequence will be referred to as the backbone sequence.

[0179] 1) Preparation of the DNA to be embedded: The plasmid DNA to be embedded (SEQ ID NO: 155) was prepared by Genscript. An amplified fragment (SEQ ID NO: 158) was prepared by PCR using primers (SEQ ID NO: 156, 157) complementary to the upstream and downstream regions of the target sequence contained in the plasmid DNA. A restriction enzyme treatment solution was prepared by restriction enzyme treatment using BciVI (New England Bio). The linear double-stranded DNA to be embedded was prepared by excising the restriction enzyme treatment solution from the agarose gel after agarose gel electrophoresis and purifying it. More specifically, the PCR reaction mixture composition was 80 μL PrimeSTAR® Max DNA Polymerase (Takara Bio), 10 pg plasmid DNA, and 0.5 μM primers, reacting in a total volume of 160 μL. After the PCR reaction, 104 μL of this mixture was mixed with 15 μL of restriction enzyme BciVI (New England Bio) and 13 μL of rCutSmart™ Buffer (New England Bio), and incubated at 37°C for 1 hour. The linear double-stranded DNA produced by this reaction had a sequence in which one base was removed from the 5' end of both strands of the double-stranded DNA complementary to SEQ ID NO: 154; in other words, it had a single overhang at the 3' end. This restriction enzyme-treated solution was subjected to electrophoresis using a 1% (w / v) agarose gel, and the DNA was excised and purified from the agarose gel. The excised gel was gel-extracted using FastGene™ Gel / PCR Extraction Kit 300prep, and finally 20 μL of linear double-stranded DNA was obtained. Plasmid DNA and linear double-stranded DNA were subjected to qPCR in the same manner as in Experimental Example 2, and their concentrations were quantified.

[0180] 2) The composition of the reaction solution for the sealing reaction and purity evaluation was 25 mM HEPES-KOH and 5 mM MgCl. ２0.5 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 5% (w / v) polyethylene glycol 6000, 163 mM NaCl, 2 nM AAV2 empty capsid, 1 nM plasmid DNA sequence or linear double-stranded DNA, 0 nM or 100 nM Rep68, pH 7.5. The reaction volume was 20 μL and reacted at 37°C for 1 hour using a thermal cycler. The target sequence and backbone sequence in the reacted sample were quantified by qPCR. The target DNA sequence was quantified using the probe method with the forward and reverse primers shown in SEQ ID NOs. 57 and 58, and the probe shown in SEQ ID NO. 59. The standard sample was plasmid DNA quantified in advance in this experiment. The backbone sequence was determined by the probe method using the forward and reverse primers shown in SEQ ID NOs. 159 and 160, and a probe modified with FAM® dye at the 5' end and TAMRA® quencher at the 3' end of the sequence shown in SEQ ID NO. 161. The standard sample was plasmid DNA that had been quantified in advance in this experiment.

[0181] Table 9 shows the concentrations (vg / μL) of the target sequence or backbone sequence in samples using plasmid DNA or linear double-stranded DNA. The DNA concentration of the detected target sequence was 1.1 × 10⁻¹⁴ when plasmid DNA was used. ４ When using vg / μL and linear double-stranded DNA, the result is 3.9 × 10⁻⁶. ６ The concentration was vg / μL, and the yield of capsids containing the target sequence was 339 times higher when linear double-stranded DNA was used. Furthermore, the concentration of backbone sequence DNA, which becomes an impurity when encapsulated in AAV capsids, was 1.7 × 10⁻⁶ when plasmid DNA was used. ４ When using vg / μL and linear double-stranded DNA, the result is 1.9 × 10⁻⁶. １ The concentration was vg / μL. The ratio of the backbone sequence to the target sequence was 146% when plasmid DNA was used as the substrate, and 0.00050% when linear double-stranded DNA was used. In other words, it was shown that AAVs containing the target sequence can be prepared with high purity by using linear double-stranded DNA as the inclusion substrate.

[0182] [Experimental Example 24] Inclusion reaction using plasmid DNA or linear double-stranded DNA without ITR sequence and comparison of the purity of the product. In Experimental Example 23, it was confirmed that when plasmid DNA was used as the inclusion sequence to inclusion DNA into a capsid, the backbone sequence was also inclusion in addition to the target sequence. In this experimental example, we compared whether plasmid DNA is inclusion or not, and if so, whether the backbone sequence is also inclusion, when the target sequence does not contain ITR, with the conditions when linear double-stranded DNA is inclusion. In this experimental example, the target sequence refers to the sequence of SEQ ID NO: 162, and the plasmid DNA sequence is the sequence shown in SEQ ID NO: 114. Hereinafter, in this experimental example, the region of the plasmid DNA sequence other than the target sequence will be referred to as the backbone sequence.

[0183] 1) Preparation of the DNA to be embedded The plasmid DNA to be embedded (SEQ ID NO: 114) was the same as described in Experimental Example 21. The DNA of the target sequence was amplified as an amplified fragment by PCR using primers (SEQ ID NOs: 163, 164) complementary to the upstream and downstream regions of the target sequence contained in the plasmid DNA. The base length of the target sequence is 2194 bp. Subsequently, linear double-stranded DNA to be embedded was prepared by excising and purifying it from the agarose gel after agarose gel electrophoresis. The PCR reaction mixture was composed of 75 μL PrimeSTAR® Max DNA Polymerase (Takara Bio), with 0.05 pg plasmid DNA and 1.0 μM of each primer, reacting in a total volume of 150 μL. This reaction mixture was subjected to electrophoresis using a 1% (w / v) agarose gel, and excision and purification were performed from the agarose gel. The excised gel was gel-extracted using FastGene® Gel / PCR Extraction Kit 300prep, ultimately yielding 30 μL of linear double-stranded DNA. The obtained DNA was quantified by qPCR using the probe method, as in Experimental Example 1. The standard sample was the sequence shown in Sequence ID No. 60.

[0184] 2) The composition of the reaction solution for the sealing reaction and purity evaluation was 25 mM HEPES-KOH and 5 mM MgCl. ２1.0 mM tris(2-carboxyethyl)phosphine, 4 mM ATP, 0.1% Pluronic® F-68, 70 mM NaCl, 1 nM AAV2 empty capsid, 1 nM plasmid DNA, or linear double-stranded DNA, 0 nM or 100 nM Rep68, pH 7.5. The reaction volume was 40 μL and reacted at 37°C for 1 hour using a thermal cycler. The target sequence and backbone sequence in the reacted sample were quantified by qPCR as in Experimental Example 23. The target sequence was quantified using the probe method with the forward and reverse primers shown in SEQ ID NOs. 57 and 58, and the probe shown in SEQ ID NO. 59. The standard sample was plasmid DNA quantified in advance in this experiment. The backbone sequence was determined by the probe method using the forward and reverse primers shown in SEQ ID NOs. 165 and 166, and a probe modified with FAM® dye at the 5' end and TAMRA® quencher at the 3' end of the sequence shown in SEQ ID NO. 167. The standard sample was plasmid DNA that had been quantified in advance in this experiment.

[0185] Table 10 shows the concentrations (vg / μL) of the target sequence or backbone sequence in samples using plasmid DNA or linear double-stranded DNA. The DNA concentration of the detected target sequence was 4.0 × 10⁶ when plasmid DNA was used. ５ When using vg / μL and linear double-stranded DNA, the result is 1.8 × 10⁻⁶. ８ The concentration was vg / μL, and the yield of capsids containing the target sequence was 449 times higher when linear double-stranded DNA was used. Furthermore, the concentration of backbone sequence DNA, which becomes an impurity when encapsulated in AAV capsids, was 6.9 × 10⁻⁶ when plasmid DNA was used. ５ When using vg / μL and linear double-stranded DNA, the result is 9.6 × 10⁻⁶. ３ The concentration was vg / μL. The ratio of the backbone sequence to the target sequence was 172% when plasmid DNA was used as the substrate, and 0.005% when linear double-stranded DNA was used. In other words, it was shown that AAV particles containing the target sequence can be prepared with high purity by using linear double-stranded DNA as the inclusion substrate.

[0186] [Experimental Example 25] Verification of the necessity of DNA cleavage activity and DNA helicase activity of Rep protein in inclusion reaction. Rep protein is a protein with multiple functions, including DNA binding activity, nicking activity that introduces single-strand breaks (nicks) into double-stranded DNA, ATPase activity, and DNA helicase activity that separates double-stranded DNA into single-stranded DNA. In this invention, we investigated which activity of the Rep protein is important for DNA inclusion activity in the capsid. It is known that in the AAV2-derived Rep68 protein, a mutant in which tyrosine at residue 156 is replaced with phenylalanine shows a significant decrease in nicking activity, and a mutant in which lysine at residue 340 is replaced with histidine shows a significant decrease in ATPase activity and DNA helicase activity. Therefore, we evaluated the inclusion activity using the Y156F mutant and K340H mutant, which were introduced into GFP-added Rep68 with these two mutations. The amino acid sequences of the Rep68 Y156F mutant and K340H mutant are those described in SEQ ID NOs. 52 and 168, respectively.

[0187] 1) Preparation of Y156F and K340H mutants Plasmids SEQ ID NOs. 169 and 170 were used to express the Y156F and K340H mutants, respectively. Using these plasmids, E. coli BL21 (DE3) strains were transformed in the same manner as in Experimental Example 1, and after inducing expression of the Rep68 mutant, affinity purification was performed to prepare the Y156F and K340H mutants. Wild-type GFP-added Rep68 without the mutation was also prepared as a positive control in the same manner as in Experimental Example 1.

[0188] 2) The composition of the reaction solution for the sealing reaction is 25 mM HEPES-KOH and 5 mM MgCl ２The reaction mixture consisted of 1.0 mM dithiothreitol, 2 mM ATP, 0.1% Pluronic® F-68, 0.2 μg / μL BSA, 1 nM AAV2 empty capsid, 1 nM DNA sequence (SEQ ID NO: 65), and 20 nM various Rep68 proteins, with a pH of 7.5. The reaction volume was 24 μL. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the reaction mixture was reacted at 37°C for 1 hour using a thermal cycler. For these samples, qPCR was performed in the same manner as in Experimental Example 2 to detect the inclusion sequences.

[0189] Figure 18 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each Rep protein species. The Y156F mutant showed a DNA sequence concentration almost equivalent to that of wild-type Rep68, confirming that the encapsulation activity was maintained. On the other hand, the K340H mutant showed a DNA sequence concentration equivalent to that of the negative control group without enzyme addition, confirming that the encapsulation activity was inactivated. In other words, it was confirmed that the DNA helicase activity and / or ATPase activity of the Rep protein are important for encapsulation in this invention.

[0190] [Experimental Example 26] Temperature study during the sealing reaction. The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl. ２ The reaction mixture consisted of 1.0 mM dithiothreitol, 2 mM ATP, 0.1% Pluronic® F-68, 65 mM NaCl, 9 nM AAV2 empty capsid, 3 nM DNA sequence (SEQ ID NO: 65), 100 nM Rep68, and pH 7.5, with a reaction volume of 24 μL. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the reaction mixture was reacted for 1 hour at 27°C, 32°C, 37°C, 41°C, or 45°C using a thermal cycler. The inclusion sequence was detected by qPCR on these samples in the same manner as in Experimental Example 2.

[0191] Figure 19 shows the concentration (vg / μL) of the DNA sequence encapsulated in the capsid for each reaction temperature. While it was confirmed that the encapsulation reaction could be carried out at any reaction temperature, the concentration of the DNA sequence increased from 27°C to 41°C, and it was confirmed that the optimal temperature was around 37°C to 41°C.

[0192] [Experimental Example 27] Investigation of combinations of PEG concentration and protein concentration 2. The composition of the reaction solution is 25 mM HEPES-KOH, 5 mM MgCl ２ The reaction mixture consisted of 5 mM dithiothreitol, 4 mM ATP, 0.05 μg / μL BSA, 0.1% Pluronic® F-68, 47.5 mM NaCl, 1 nM AAV2 empty capsid, 1 nM DNA sequence (blunt-ended sequence: SEQ ID NO: 65), 0-15% (w / v) polyethylene glycol 6,000 (PEG6,000, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog no. 169-09125), 0-30 nM Rep68, pH 7.5, and 20 μL of reaction solution. The DNA inclusion reaction into the capsid was carried out in the same manner as in Experimental Example 1, except that the above reaction mixture was reacted for 1 hour at 37°C using a thermal cycler. The inclusion sequence was detected by qPCR on these samples in the same manner as in Experimental Example 2.

[0193] Table 11 shows the concentration of the DNA sequence encapsulated in the capsid (vg / μL) for each combination of PEG concentration and Rep concentration. Similar to Experimental Example 12, the addition of PEG 6,000 increased the concentration of DNA encapsulated in the capsid compared to the non-addition condition in all Rep concentration conditions. Therefore, it was clear that the reactivity was improved by adding PEG.

Claims

1. A method for producing recombinant adeno-associated virus particles, comprising the steps of: mixing a capsid synthesized in a first system, a Rep protein synthesized in a second system different from the first system, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid.

2. A method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, thereby encapsulating the polynucleotide in the capsid, wherein the composition resulting from the mixing in the step substantially consists only of a capsid and a Rep protein as proteins.

3. A method for producing recombinant adeno-associated virus particles, comprising the steps of mixing a capsid, a Rep protein, and a polynucleotide encoding a target sequence, and encapsulating the polynucleotide in the capsid, wherein the composition produced by the mixing in the step does not contain E1A, E1B, E2A, E4, and VA.

4. The manufacturing method according to any one of claims 1 to 3, wherein the capsid is purified.

5. The manufacturing method according to any one of claims 1 to 3, wherein the Rep protein is purified.

6. The manufacturing method according to any one of claims 1 to 3, wherein the polynucleotide is linear.

7. The manufacturing method according to claim 6, wherein the polynucleotide is linear and double-stranded.

8. The manufacturing method according to any one of claims 1 to 3, wherein the length of the polynucleotide is 20 to 5,000 bases.

9. The method for producing a polynucleotide according to any one of claims 1 to 3, wherein the polynucleotide includes a modified residue.

10. The manufacturing method according to any one of claims 1 to 3, wherein the polynucleotide does not contain part or all of the ITR.

11. The manufacturing method according to any one of claims 1 to 3, wherein the polynucleotide comprises RNA.

12. The manufacturing method according to claim 1, wherein the first system uses first cells that express a capsid.

13. The manufacturing method according to claim 1, wherein the second system uses a second cell that expresses the Rep protein.

14. The manufacturing method according to any one of claims 1 to 3, wherein the Rep protein is one or more selected from Rep78, Rep68, Rep52, and Rep40.

15. The manufacturing method according to any one of claims 1 to 3, wherein the Rep protein is one or more selected from Rep78 and Rep68.

16. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 1 pM or more and 1 μM or less.

17. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 10 pM or more and 100 nM or less.

18. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 100 pM or more and 50 nM or less.

19. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the capsid is mixed so that the concentration of the capsid is 1 nM or more and 20 nM or less.

20. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 1 pM or more and 100 μM or less.

21. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 10 pM or more and 10 μM or less.

22. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 1 nM or more and 1 μM or less.

23. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the Rep protein is mixed so that the concentration of the Rep protein is 10 nM or more and 100 nM or less.

24. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the polynucleotides are mixed so that the concentration of the polynucleotides is 1 pM or more and 1 μM or less.

25. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the polynucleotides are mixed so that the concentration of the polynucleotides is 10 pM or more and 100 nM or less.

26. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the polynucleotides are mixed so that the concentration of the polynucleotides is 100 pM or more and 50 nM or less.

27. The manufacturing method according to any one of claims 1 to 3, wherein in the encapsulation step, the polynucleotides are mixed so that the concentration of the polynucleotides is 1 nM or more and 20 nM or less.

28. The manufacturing method according to any one of claims 1 to 3, wherein a nonionic water-soluble polymer is further mixed in the encapsulation step.

29. The manufacturing method according to claim 28, wherein the weight-average molecular weight of the water-soluble polymer is 600 to 4,000,000.

30. The manufacturing method according to claim 28, wherein the water-soluble polymer is one or more selected from the group consisting of polyether-based water-soluble polymers, water-soluble polysaccharides, vinyl-based water-soluble polymers, and sucrose-epichlorohydrin copolymers.

31. The method for producing a water-soluble polymer according to claim 28, wherein the water-soluble polymer is one or more selected from the group consisting of polyethylene glycol (PEG), poly(propylene glycol), dextran, methylcellulose, polyvinylpyrrolidone, and sucrose-epichlorohydrin copolymer.

32. The manufacturing method according to claim 28, wherein in the encapsulation step, the water-soluble polymer is mixed so that the concentration of the water-soluble polymer is 0.03 to 10% (w / v).

33. The manufacturing method according to any one of claims 1 to 3, wherein a surfactant is further mixed in the encapsulation step.

34. The manufacturing method according to claim 33, wherein the surfactant is a nonionic surfactant.

35. The manufacturing method according to claim 33, wherein the surfactant has a polyoxyethylene chain.

36. The manufacturing method according to claim 33, wherein in the encapsulation step, the surfactant is mixed so that the concentration of the surfactant is 0.0001 to 3% (w / v).

37. The manufacturing method according to any one of claims 1 to 3, wherein the sealing step is performed at 20 to 55°C.

38. The manufacturing method according to claim 2, wherein the composition does not contain E1A, E1B, E2A, E4, and VA.

39. The manufacturing method according to claim 2, 3, or 38, wherein the composition substantially does not contain cell lysate.

40. Recombinant adeno-associated virus particles in which the capsid contains a) modified residues, b) some or all of the ITR, or c) a polynucleotide containing RNA.

41. The recombinant adeno-associated virus particle according to claim 40, wherein the polynucleotide is linear.

42. The recombinant adeno-associated virus particle according to claim 40 or 41, wherein the polynucleotide comprises a modified residue.

43. A composition comprising a capsid, a rep protein, and a) modified residues, b) some or all of the ITR, or c) a polynucleotide containing RNA, and substantially free from cell disruption solution.

44. The composition according to claim 43, wherein the capsid is purified.

45. The composition according to claim 43 or 44, wherein the Rep protein is purified.

46. ​​The composition according to claim 43 or 44, wherein the Rep protein is one or more selected from Rep78, Rep68, Rep52, and Rep40.

47. The composition according to claim 43 or 44, wherein the polynucleotide is linear.

48. The composition according to claim 43 or 44, wherein the polynucleotide comprises a modified residue.

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